title: PeerJ description: Articles published in PeerJ link: https://peerj.com/articles/index.rss3?journal=peerj&month=2021-09 creator: info@peerj.com PeerJ errorsTo: info@peerj.com PeerJ language: en title: Identifying optimal bioinformatics protocols for aerosol microbial community data link: https://peerj.com/articles/12065 last-modified: 2021-09-30 description: Microbes are fundamental to Earth’s ecosystems, thus understanding ecosystem connectivity through microbial dispersal is key to predicting future ecosystem changes in a warming world. However, aerial microbial dispersal remains poorly understood. Few studies have been performed on bioaerosols (microorganisms and biological fragments suspended in the atmosphere), despite them harboring pathogens and allergens. Most environmental microbes grow poorly in culture, therefore molecular approaches are required to characterize aerial diversity. Bioinformatic tools are needed for processing the next generation sequencing (NGS) data generated from these molecular approaches; however, there are numerous options and choices in the process. These choices can markedly affect key aspects of the data output including relative abundances, diversity, and taxonomy. Bioaerosol samples have relatively little DNA, and often contain novel and proportionally high levels of contaminant organisms, that are difficult to identify. Therefore, bioinformatics choices are of crucial importance. A bioaerosol dataset for bacteria and fungi based on the 16S rRNA gene (16S) and internal transcribed spacer (ITS) DNA sequencing from parks in the metropolitan area of Auckland, Aotearoa New Zealand was used to develop a process for determining the bioinformatics pipeline that would maximize the data amount and quality generated. Two popular tools (Dada2 and USEARCH) were compared for amplicon sequence variant (ASV) inference and generation of an ASV table. A scorecard was created and used to assess multiple outputs and make systematic choices about the most suitable option. The read number and ASVs were assessed, alpha diversity was calculated (Hill numbers), beta diversity (Bray–Curtis distances), differential abundance by site and consistency of ASVs were considered. USEARCH was selected, due to higher consistency in ASVs identified and greater read counts. Taxonomic assignment is highly dependent on the taxonomic database used. Two popular taxonomy databases were compared in terms of number and confidence of assignments, and a combined approach developed that uses information in both databases to maximize the number and confidence of taxonomic assignments. This approach increased the assignment rate by 12–15%, depending on amplicon and the overall assignment was 77% for bacteria and 47% for fungi. Assessment of decontamination using “decontam” and “microDecon” was performed, based on review of ASVs identified as contaminants by each and consideration of the probability of them being legitimate members of the bioaerosol community. For this example, “microDecon’s” subtraction approach for removing background contamination was selected. This study demonstrates a systematic approach to determining the optimal bioinformatics pipeline using a multi-criteria scorecard for microbial bioaerosol data. Example code in the R environment for this data processing pipeline is provided. creator: Katie Miaow creator: Donnabella Lacap-Bugler creator: Hannah L. Buckley uri: https://doi.org/10.7717/peerj.12065 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Miaow et al. title: Blockchain for genomics and healthcare: a literature review, current status, classification and open issues link: https://peerj.com/articles/12130 last-modified: 2021-09-30 description: The tremendous boost in the next generation sequencing technologies and in the “omics” technologies resulted in the generation of hundreds of gigabytes of data per day. Nowadays, via integrating -omics data with other data types, such as imaging and electronic health record (EHR) data, panomics studies attempt to identify novel and potentially actionable biomarkers for personalized medicine applications. In this respect, for the accurate analysis of -omics data and EHR, there is a need to establish secure and robust pipelines that take the ethical aspects into consideration, regulate privacy and ownership issues, and data sharing. These days, blockchain technology has picked up significant attention in diverse fields, including genomics, since it offers a new solution for these problems from a different perspective. Blockchain is an immutable transaction ledger, which offers secure and distributed system without a central authority. Within the system, each transaction can be expressed with cryptographically signed blocks, and the verification of transactions is performed by the users of the network. In this review, firstly, we aim to highlight the challenges of EHR and genomic data sharing. Secondly, we attempt to answer “Why” or “Why not” the blockchain technology is suitable for genomics and healthcare applications in detail. Thirdly, we elucidate the general blockchain structure based on the Ethereum, which is a more suitable technology for the genomic data sharing platforms. Fourthly, we review current blockchain-based EHR and genomic data sharing platforms, evaluate the advantages and disadvantages of these applications, and classify these applications using different metrics. Finally, we conclude by discussing the open issues and introducing our suggestion on the topic. In summary, to facilitate the diagnosis, monitoring and therapy of diseases with the effective analysis of -omics data with other available data types, through this review, we put forward the possible implications of the blockchain technology to life sciences and healthcare. creator: Beyhan Adanur Dedeturk creator: Ahmet Soran creator: Burcu Bakir-Gungor uri: https://doi.org/10.7717/peerj.12130 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Adanur Dedeturk et al. title: Dynamic changes in moisture content and applicability analysis of a typical litter prediction model in Yunnan Province link: https://peerj.com/articles/12206 last-modified: 2021-09-30 description: BackgroundForest fire risk predictions are based on the most conservation daily predictions, and the lowest litter moisture content of each day is often used to predict the day’s fire risk. Yunnan Province is the area with the most frequent and serious forest fires in China, but there is almost no research on the dynamic changes and model predictions of the litter moisture content in this area. Therefore, to reduce the occurrence of forest fires and improve the accuracy of forest fire risk predictions, it is necessary to understand these dynamic changes and establish an appropriate prediction model for the typical litter moisture content in Yunnan Province.MethodDuring the fire prevention period, daily dynamic changes in the litter moisture content are obtained by monitoring the daily step size, and the relationships between the litter moisture content and meteorological elements are analyzed. In this study, the meteorological element regression method, moisture code method and direction estimation method are selected to establish litter moisture content prediction models, and the applicability of each model is analyzed.ResultsWe found that dynamic changes in the litter moisture content have obvious lags compared with meteorological elements, and the litter moisture content is mainly related to the air temperature, relative humidity and wind speed. With an increase in the sampling interval of meteorological elements, the significances of these correlations first increase and then decrease. The moisture content value obtained by directly using the moisture code method in the Fire Weather Index (FWI) significantly different from the measured value, so this method is not applicable. The mean absolute error (MAE) and mean relative error (MRE) values obtained with the meteorological element regression method are 2.97% and 14.06%, those from the moisture code method are 3.27% and 14.07%, and those from the direct estimation method are 2.82% and 12.76%, respectively.ConclusionsThe direct estimation method has the lowest error and the strongest extrapolation ability; this method can meet the needs of daily fire forecasting. Therefore, it is feasible to use the direct estimation method to predict litter moisture contents in Yunnan Province. creator: Yunlin Zhang creator: Lingling Tian uri: https://doi.org/10.7717/peerj.12206 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Zhang and Tian title: Mandible histology in Metoposaurus krasiejowensis (Temnospondyli, Stereospondyli) from the Upper Triassic of Poland link: https://peerj.com/articles/12218 last-modified: 2021-09-30 description: Recent studies that have systematically augmented our knowledge of dermal bones of the Late Triassic temnospondyl amphibian Metoposaurus krasiejowensis have mostly focused on shoulder girdle elements and the skull. So far, histological data on the mandible are still scant. For the present study, two mandibles have been examined, using 50 standard thin sections. Dermal bones of the mandible reveal a uniform diploë structure, with the external cortex consisting of moderately vascularised, parallel-fibred bone, as well as a distinct alternation of thick zones and thinner annuli. Dense bundles of well-mineralised Sharpey’s fibres are seen in the external cortex over the entire length of the mandible. The trabecular middle region is highly porous and well vascularised, showing small primary vascular canals and more numerous secondary osteons; irregular erosion spaces occur in large numbers as well. The thin and poorly vascular internal cortex consists of parallel-fibred bone. The articular is not a dermal bone in origin, having been formed of a thin layer of avascular cortex and a very extensive, trabecular middle region. In contrast to the dermal bones of the mandible, the articular developed from a cartilaginous precursor, as evidenced by numerous remains of calcified cartilage in the central parts of the bone. Histological variability is extremely high along the mandible, its anterior part being characterised by high compactness and biomechanically good resistance in contrast to the highly porous posterior parts. Distinct variations of bone thickness and degree of bone porosity in specific areas of the mandible, may be due to local differences in biomechanics during feeding. The microstructure of the mandible corroborates a previous study of the active and ambush predation strategy in metoposaurids. creator: Kamil Gruntmejer creator: Adam Bodzioch creator: Dorota Konietzko-Meier uri: https://doi.org/10.7717/peerj.12218 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Gruntmejer et al. title: Elevated serum FGF21 predicts the major adverse cardiovascular events in STEMI patients after emergency percutaneous coronary intervention link: https://peerj.com/articles/12235 last-modified: 2021-09-30 description: BackgroundAlthough there have been several studies related to serum fibroblast growth factor 21 (FGF21) levels and acute myocardial infarction, the value of serum FGF21 levels in ST-segment elevation myocardial infarction (STEMI) patients after emergency percutaneous coronary intervention (PCI) has not been previously investigated.MethodsA total of 348 STEMI patients who underwent emergency PCI were enrolled from January 2016 to December 2018. The primary endpoint was the occurrence of major adverse cardiovascular events (MACEs), with a median follow-up of 24 months. Eighty patients with stable angina (SA) who underwent selective PCI served as the control group. Serum FGF21 levels were measured by ELISA.ResultsSerum FGF21 levels were significantly higher in the STEMI group than in the SA group (225.03 ± 37.98 vs. 135.51 ±  34.48, P < 0.001). Multiple linear regression analysis revealed that serum FGF21 levels were correlated with NT-proBNP (P < 0.001). According to receiver operating characteristic (ROC) analysis, the areas under the ROC curve (AUCs) of FGF21 and NT-proBNP were 0.812 and 0.865, respectively. The Kaplan-Meier curves showed that STEMI patients with lower FGF21 levels had an increased MACE-free survival rate. Cox analysis revealed that high FGF21 levels (HR: 2.011, 95% CI: [1.160–3.489]) proved to be a powerful tool in predicting the risk of MACEs among STEMI patients after emergency PCI.ConclusionElevated FGF21 levels on admission have been shown to be a powerful predictor of MACEs for STEMI patients after emergency PCI. creator: Lingyun Gu creator: Wenlong Jiang creator: Huidong Qian creator: Ruolong Zheng creator: Weizhang Li uri: https://doi.org/10.7717/peerj.12235 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Gu et al. title: Turf performance and physiological responses of native Poa species to summer stress in Northeast China link: https://peerj.com/articles/12252 last-modified: 2021-09-30 description: Rapid rise in temperature in summer causes severe injury to cool-season turfgrass of both native species and introduced ones in Heilongjiang of Northeast China. The objectives of this study were to compare physiological responses to seasonal heat stresses and turf performances between native and introduced commercial Poa accessions. Three Chinese native Poa species (i.e., P. pratensis, P. sibirica and P. sphondylodes) and three USA Kentucky bluegrass cultivars (ie. ‘Midnight’, ‘Moonlight’ and ‘BlueChip’) were evaluated under field conditions in 2017 and 2018. All accessions showed unique characteristics and considerable seasonal differences in response to temperatures. However, performances over all accessions were largely similar in early spring and autumn. In summer, native P. pratensis performed similar to ‘Midnight’, ‘Moonlight’ or ‘BlueChip’, with respect to such traits or parameters as quality, coverage, color intensity, growth rate, osmolytes, ROS and anti-oxidant production. Native P. pratensis could be used as a new turf resource for further improvement and application under the specific climatic conditions in Heilongjiang; native P. sphondylodes may be used in repairing damaged environments or for alternative seasonal greenness. creator: Yajun Chen creator: Zhixin Guo creator: Lili Dong creator: Zhenxuan Fu creator: Qianjiao Zheng creator: Gaoyun Zhang creator: Ligang Qin creator: Xiaoyang Sun creator: Zhenjie Shi creator: Shah Fahad creator: Fuchun Xie creator: Shah Saud uri: https://doi.org/10.7717/peerj.12252 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Chen et al. title: Highly comparable metabarcoding results from MGI-Tech and Illumina sequencing platforms link: https://peerj.com/articles/12254 last-modified: 2021-09-30 description: With the developments in DNA nanoball sequencing technologies and the emergence of new platforms, there is an increasing interest in their performance in comparison with the widely used sequencing-by-synthesis methods. Here, we test the consistency of metabarcoding results from DNBSEQ-G400RS (DNA nanoball sequencing platform by MGI-Tech) and NovaSeq 6000 (sequencing-by-synthesis platform by Illumina) platforms using technical replicates of DNA libraries that consist of COI gene amplicons from 120 soil DNA samples. By subjecting raw sequencing data from both platforms to a uniform bioinformatics processing, we found that the proportion of high-quality reads passing through the filtering steps was similar in both datasets. Per-sample operational taxonomic unit (OTU) and amplicon sequence variant (ASV) richness patterns were highly correlated, but sequencing data from DNBSEQ-G400RS harbored a higher number of OTUs. This may be related to the lower dominance of most common OTUs in DNBSEQ data set (thus revealing higher richness by detecting rare taxa) and/or to a lower effective read quality leading to generation of spurious OTUs. However, there was no statistical difference in the ASV and post-clustered ASV richness between platforms, suggesting that additional denoising step in the ASV workflow had effectively removed the ‘noisy’ reads. Both OTU-based and ASV-based composition were strongly correlated between the sequencing platforms, with essentially interchangeable results. Therefore, we conclude that DNBSEQ-G400RS and NovaSeq 6000 are both equally efficient high-throughput sequencing platforms to be utilized in studies aiming to apply the metabarcoding approach, but the main benefit of the former is related to lower sequencing cost. creator: Sten Anslan creator: Vladimir Mikryukov creator: Kęstutis Armolaitis creator: Jelena Ankuda creator: Dagnija Lazdina creator: Kristaps Makovskis creator: Lars Vesterdal creator: Inger Kappel Schmidt creator: Leho Tedersoo uri: https://doi.org/10.7717/peerj.12254 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Anslan et al. title: Discordance in maternal and paternal genetic markers in lesser long-nosed bat Leptonycteris yerbabuenae, a migratory bat: recent expansion to the North and male phylopatry link: https://peerj.com/articles/12168 last-modified: 2021-09-29 description: Leptonycteris yerbabuenae, the lesser long-nosed bat is an abundant migratory nectar-feeding bat found in most of Mexico, and in some areas of northern Central America and small sections of southwestern USA. We analyzed the distribution of the maternal and paternal lineages of this species with phylogeographic methods based on two mitochondrial markers, Cyt-b and D-loop, and a marker located in the Y chromosome, DBY. We obtained tissue samples from 220 individuals from 23 localities. Levels of genetic diversity (haplotype diversity, Hd) were high (Cyt-b = 0.757; D-loop = 0.8082; DBY = 0.9137). No clear patterns of population genetic structure were found for mitochondrial markers, while male genetic differentiation suggested the presence of two lineages: one from Mexican Pacific coast states and another from central-southern Mexico; in accordance to strong male philopatry and higher female migration. We used genealogical reconstructions based on Bayesian tools to calculate divergence times, and to test coalescent models to explain changes in L. yerbabuenae historical demography. Our results show that recent demographic changes were consistent with global climatic changes (∼130,000 kyr ago for Cyt-b and ∼160,000 kyr for D-loop) and divergence times dated from molecular genealogies exhibited older divergence times, Cyt-b (4.03 mya), D-loop (10.26 mya) and DBY (12.23 mya). Accordingly, the female lineage underwent demographic expansion associated to Pleistocene climate change, whereas the male lineage remained constant. creator: Roberto-Emiliano Trejo-Salazar creator: Gabriela Castellanos-Morales creator: DulceCarolina Hernández-Rosales creator: Niza Gámez creator: Jaime Gasca-Pineda creator: Miguel Rene Morales Garza creator: Rodrigo Medellin creator: Luis E. Eguiarte uri: https://doi.org/10.7717/peerj.12168 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Trejo-Salazar et al. title: Evaluating changes in student health, wellbeing and social circumstances before and during COVID-19 pandemic restrictions in Australia link: https://peerj.com/articles/12078 last-modified: 2021-09-29 description: The impacts of COVID-19 have been felt on a global scale, with associated physical distancing restrictions and economic downturn having flow-on effects for mental health and wellbeing across the community, and for university students in particular. First-year pharmaceutical and medical science students completing a common introductory population health course at an Australian university are routinely surveyed at the beginning of the semester as part of the course. Survey data inform teaching approaches based on understanding the ‘real life’ commitments and health profiles of students, and deidentified data form part of the teaching material. The 2020 student cohort was invited to complete a second follow-up survey during COVID-19 physical distancing restrictions. A total of n = 126 students completed both the initial and follow-up surveys (50.6% response rate), and n = 99 (39.8% of the total cohort) consented for their data to be included in research. There was a non-significant decrease in student employment; however, 22% of all students reported loss of work due to COVID-19. There was a statistically significant decrease in the proportion of students undertaking sufficient levels of physical activity, and a statistically significant increase in reported family stressors associated with loss of employment or an inability to gain employment between March and May 2020. Two-thirds of respondents reported increased stress as an impact of the transition to online learning. Implementation of holistic strategies, incorporating attention to additional factors influencing students’ capacity to engage in study, and which may have long-term impacts across the life of the degree program, should be considered. creator: Dannielle Post creator: Agnes Vitry creator: Katherine L. Baldock uri: https://doi.org/10.7717/peerj.12078 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Post et al. title: Anthropogenic transformations of river ecosystems are not always bad for the environment: Multi-taxa analyses of changes in aquatic and terrestrial environments after dredging of a small lowland river link: https://peerj.com/articles/12224 last-modified: 2021-09-29 description: Rivers are one of the most commonly transformed aquatic ecosystems. Most papers present significantly negative effects of activities such as dredging or channel regulation on the ecological status of rivers. The purpose of this work was to compare the response of various groups of invertebrates (Mollusca, Hydrachnidia, Odonata, Heteroptera, Coleoptera and Trichoptera) to an intervention involving dredging in conjunction with the removal of riparian vegetation. Habitat diversity increased after the dredging, and more individuals and species were caught than before the dredging. The increase in habitat diversity after the dredging translated into an increase in the species diversity of most investigated groups. Individual groups of invertebrates showed varied responses to the dredging, depending on the role of the terrestrial phase in their life cycle: the greater the role of the terrestrial phase in the life cycle, the more the group was affected by changes in the terrestrial environment following the intervention. In consequence, the intervention had the greatest negative impact on insects, and among these, on adult Odonata. The following conclusions can be drawn: (1) Dredging can benefit a previously anthropogenically transformed river ecosystem by increasing habitat diversity; (2) Odonata are particularly useful for assessing the impact of this type of intervention on invertebrate communities. They can be considered good indicators of habitat disturbances in both aquatic and terrestrial ecosystems. creator: Robert Stryjecki creator: Andrzej Zawal creator: Tomasz Krepski creator: Edyta Stępień creator: Edyta Buczyńska creator: Paweł Buczyński creator: Stanisław Czachorowski creator: Łukasz Jankowiak creator: Joanna Pakulnicka creator: Anna Sulikowska-Drozd creator: Vladimir Pešić creator: Grzegorz Michoński creator: Michał Grabowski creator: Aleksandra Jabłońska creator: Magdalena Achrem creator: Tomasz Olechwir creator: Lech Pietrzak creator: Agnieszka Szlauer-Łukaszewska uri: https://doi.org/10.7717/peerj.12224 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Stryjecki et al. title: The oldest species of Peltoperleidus (Louwoichthyiformes, Neopterygii) from the Middle Triassic (Anisian) of China, with phylogenetic and biogeographic implications link: https://peerj.com/articles/12225 last-modified: 2021-09-29 description: The previously alleged ‘perleidid’ genus Peltoperleidus is a stem-neopterygian fish taxon with two or three horizontal rows of notably deepened flank scales. Until recently, members of this genus were known only from the Ladinian (late Middle Triassic) or near the Anisian/Ladinian boundary (~242 Ma) in southern Switzerland and northern Italy. Here, I report the discovery of a new species of the genus, Peltoperleidus asiaticus sp. nov., based on three well-preserved specimens from the Anisian (early Middle Triassic, ~244 Ma) of Luoping, eastern Yunnan, China. The discovery extends the geological range of Peltoperleidus by approximately two million years and documents the first record of the genus in Asia. Similar to its relatives (represented by P. macrodontus) from Europe, P. asiaticus sp. nov. is likely a small-sized durophagous predator with dentition combining grasping and crushing morphologies. Results of a cladistic analysis unite four species of Peltoperleidus as a monophyletic group within the Louwoichthyiformes, and suggest that the presence of two horizontal rows of notably deepened scales was independently evolved in Peltoperleidus and another stem-neopterygian taxon Altisolepis. P. asiaticus sp. nov. is nested at the base of Peltoperleidus, and a new family Peltoperleididae is proposed for the genus, contrasting the previous placement of Peltoperleidus in the poorly defined, paraphyletic ‘Perleididae’. Comparative studies of the basal peltoperleidid from China with its younger relatives from Europe provide new insights into the evolutionary origin and paleogeographic distribution of this clade. creator: Guang-Hui Xu uri: https://doi.org/10.7717/peerj.12225 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Xu title: Nickel stress-tolerance in plant-bacterial associations link: https://peerj.com/articles/12230 last-modified: 2021-09-29 description: Nickel (Ni) is an essential element for plant growth and is a constituent of several metalloenzymes, such as urease, Ni-Fe hydrogenase, Ni-superoxide dismutase. However, in high concentrations, Ni is toxic and hazardous to plants, humans and animals. High levels of Ni inhibit plant germination, reduce chlorophyll content, and cause osmotic imbalance and oxidative stress. Sustainable plant-bacterial native associations are formed under Ni-stress, such as Ni hyperaccumulator plants and rhizobacteria showed tolerance to high levels of Ni. Both partners (plants and bacteria) are capable to reduce the Ni toxicity and developed different mechanisms and strategies which they manifest in plant-bacterial associations. In addition to physical barriers, such as plants cell walls, thick cuticles and trichomes, which reduce the elevated levels of Ni entrance, plants are mitigating the Ni toxicity using their own antioxidant defense mechanisms including enzymes and other antioxidants. Bacteria in its turn effectively protect plants from Ni stress and can be used in phytoremediation. PGPR (plant growth promotion rhizobacteria) possess various mechanisms of biological protection of plants at both whole population and single cell levels. In this review, we highlighted the current understanding of the bacterial induced protective mechanisms in plant-bacterial associations under Ni stress. creator: Veronika Pishchik creator: Galina Mirskaya creator: Elena Chizhevskaya creator: Vladimir Chebotar creator: Debasis Chakrabarty uri: https://doi.org/10.7717/peerj.12230 license: https://creativecommons.org/publicdomain/zero/1.0/ rights: ©2021 Pishchik et al. title: Integrative analysis of miRNA–mRNA network in idiopathic membranous nephropathy by bioinformatics analysis link: https://peerj.com/articles/12271 last-modified: 2021-09-29 description: BackgroundCurrently, several specific antigens, M-type receptor for secretory phospholipase A2(PLA2R1), thrombospondin type-1 domain-containing 7A(THSD7A), and neural epidermal growth factor-like 1 protein (NELL-1), are discovered associated with the onset of idiopathic membranous nephropathy (IMN). But the pathomechanisms of IMN still need to be further claried. Understanding the mechanisms of IMN is required to improve its diagnosis and treatment.MethodsIn this study, we constructed miRNA regulatory networks to investigate IMN development. Moreover, miRNAs and mRNAs that were differentially expressed between Idiopathic Membranous Nephropathy (IMN) patients and normal controls were examined using the GSE115857 dataset and our previous sequence study. DE miRNA target genes were determined based on the FUNRICH software, starBase, miRDB, and miRWalk, and an miRNA-mRNA network was designed using DE-mRNAs that were negatively correlated with DE-miRNAs. The miRNA-mRNA network contained 228 miRNA-mRNA pairs. Thereafter, we conducted KEGG pathway, GO functional annotation, immune-related gene screening, protein interaction networks, and potential hub gene analyses. Furthermore, 10 miRNAs and 10 genes were determined and preliminarily validated using the validation dataset from GEO. Finally, we identified which pair may offer more accurate diagnosis and therapeutic targets for IMN.ResultsTwo miRNA-mRNA pairs, miR-155-5p-FOS and miR-146a-5p-BTG2, were differentially expressed in IMN, indicating that these genes may affect IMN through immune processes. These findings may offer more accurate diagnoses and therapeutic targets for IMN. creator: Wenfang He creator: Jinshi Zhang creator: Shizhu Yuan creator: Mingzhu Liang creator: Weidong Chen creator: Juan Jin uri: https://doi.org/10.7717/peerj.12271 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 He et al. title: Phylogenetic relationships of the genus Mischonyx Bertkau, 1880, with taxonomic changes and three new species description (Opiliones: Gonyleptidae) link: https://peerj.com/articles/11682 last-modified: 2021-09-28 description: The type species of Mischonyx Bertkau 1880, Mischonyx squalidus, was described based on a juvenile. The holotype is lost. Based on a revision of publications, the genus includes 12 species, all in Brazil. The objectives of this research are: to propose a phylogenetic hypothesis for Mischonyx based on Total Evidence (TE); propose taxonomic changes based on the phylogeny; and analyze the phylogenetic hypothesis biogeographically. Using the exemplar approach to taxon selection, we studied 54 specimens, 15 outgroups and 39 ingroup taxa using seven molecular markers (28S, 12S and 16S ribosomal genes, citochrome oxidase subunit I gene, carbamoyl-phosphate synthetase gene, internal transcribed spacer subunit 2 and histone H3 gene), totaling 3,742 bp, and 128 morphological characters. We analyzed the dataset under three optimality criteria: Maximum likelihood (ML), Maximum parsimony (MP) and Bayesian. We discuss the transformation of character states throughout the phylogeny, the different phylogenetic hypotheses using different datasets and the congruence of evidence between the clades obtained by the phylogenetic analysis and the biogeographical hypothesis for the Atlantic Forest areas of endemism. We estimate that Mischonyx clade diverged 50.53 Mya, and inside the genus there are two major clades. One of them cointains species from Paraná, Santa Catarina, South of São Paulo and Serra do Mar Areas of Endemism and the other has species from Espinhaço, Bocaina, South coast of Rio de Janeiro and Serra dos Órgãos Areas of Endemism. The first split inside these two clades occurred at 48.94 and 44.80 Mya, respectively. We describe three new species from Brazil: Mischonyx minimussp. nov. (type locality: Petrópolis, Rio de Janeiro), Mischonyx intervalensissp. nov. (type locality: Ribeirão Grande, São Paulo) and Mischonyx tinguaensissp. nov (type locality: Nova Iguaçu, Rio de Janeiro). The genus Urodiabunus Mello-Leitão, 1935 is considered a junior synonym of Mischonyx. Weyhia spinifronsMello-Leitão, 1923; Weyhia clavifemur Mello-Leitão, 1927 and Geraeocormobius reitzi Vasconcelos, 2005 were transferred to Mischonyx. Mischonyx cuspidatus (Roewer, 1913) is a junior synonym of M. squalidus Bertkau, 1880. In the results of the phylogenetic analyses, Gonyleptes antiquusMello-Leitão, 1934 (former Mischonyx antiquus) does not belong in Mischonyx and its original combination is re-established. As it is now defined, Mischonyx comprises 17 species, with seven new combinations. creator: Caio Gueratto creator: Alípio Benedetti creator: Ricardo Pinto-da-Rocha uri: https://doi.org/10.7717/peerj.11682 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Gueratto et al. title: The light’s in my eyes: optical modeling demonstrates wind is more important than sea surface-reflected sunlight for foraging herons link: https://peerj.com/articles/12006 last-modified: 2021-09-28 description: Multiple lineages of birds have independently evolved foraging strategies that involve catching aquatic prey by striking at them through the water’s surface. Diurnal, visual predators that hunt across the air-water interface encounter several visual challenges, including sun glint, or reflection of sunlight by the water surface. Intense sun glint is common at the air-water interface, and it obscures visual cues from submerged prey. Visually-hunting, cross-media predators must therefore solve the problem of glint to hunt effectively. One obvious solution is to turn away from the sun, which would result in reduction of glint effects. However, turning too far will cast shadows over prey, causing them to flee. Therefore, we hypothesized that foraging herons would orient away from, but not directly opposite to the sun. Our ability to understand how predators achieve a solution to glint is limited by our ability to quantify the amount of glint that free-living predators are actually exposed to under different light conditions. Herons (Ardea spp.) are a good model system for answering questions about cross-media hunting because they are conspicuous, widely distributed, and forage throughout a variety of aquatic habitats, on a variety of submerged prey. To test our hypothesis, we employed radiative transfer modeling of water surface reflectance, drawn from optical oceanography, in a novel context to estimate the visual exposure to glint of free-living, actively foraging herons. We found evidence that Ardea spp. do not use body orientation to compensate for sun glint while foraging and therefore they must have some other, not yet understood, means of compensation, either anatomical or behavioral. Instead of facing away from the sun, herons tended to adjust their position to face into the wind at higher wind speeds. We suggest that radiative transfer modeling is a promising tool for elucidating the ecology and evolution of air-to-water foraging systems. creator: Holly K.M. Brown creator: Margaret Rubega creator: Heidi M. Dierssen uri: https://doi.org/10.7717/peerj.12006 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Milton Brown et al. title: Cryptocentrus steinhardti (Actinopterygii; Gobiidae): a new species of shrimp-goby, and a new invasive to the Mediterranean Sea link: https://peerj.com/articles/12136 last-modified: 2021-09-28 description: A new species of shrimp-goby was collected at depths of 60–80 m off the southern Israeli Mediterranean coast. A unique ‘DNA barcoding’ signature (mtDNA COI and Cytb) revealed that it differs from any other previously bar-coded goby species clustered phylogenetically with the shrimp-gobies group, in which Cryptocentrus is the most speciose genus. A morphological study supported the assignment of the fish to Cryptocentrus and differentiated the new species from its congeners. The species is described here as Cryptocentrus steinhardti n. sp. However, the present phylogenetic analysis demonstrates a paraphyly of Cryptocentrus and emphasizes the need for revision of the genus based on integrating morphological and genetic characteristics. This finding constitutes the third record of an invasive shrimp goby in the Mediterranean Sea. An intriguing ecological issue arises regarding the possible formation of a fish-shrimp symbiosis in a newly invaded territory. Describing an alien tropical species in the Mediterranean prior to its discovery in native distribution is an unusual event, although not the first such case. Several similar examples are provided in the present article. creator: Menachem Goren creator: Nir Stern uri: https://doi.org/10.7717/peerj.12136 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Goren and Stern title: The clinical diagnosis of Achilles tendinopathy: a scoping review link: https://peerj.com/articles/12166 last-modified: 2021-09-28 description: BackgroundAchilles tendinopathy describes the clinical presentation of pain localised to the Achilles tendon and associated loss of function with tendon loading activities. However, clinicians display differing approaches to the diagnosis of Achilles tendinopathy due to inconsistency in the clinical terminology, an evolving understanding of the pathophysiology, and the lack of consensus on clinical tests which could be considered the gold standard for diagnosing Achilles tendinopathy. The primary aim of this scoping review is to provide a method for clinically diagnosing Achilles tendinopathy that aligns with the nine core health domains.MethodologyA scoping review was conducted to synthesise available evidence on the clinical diagnosis and clinical outcome measures of Achilles tendinopathy. Extracted data included author, year of publication, participant characteristics, methods for diagnosing Achilles tendinopathy and outcome measures.ResultsA total of 159 articles were included in this scoping review. The most commonly used subjective measure was self-reported location of pain, while additional measures included pain with tendon loading activity, duration of symptoms and tendon stiffness. The most commonly identified objective clinical test for Achilles tendinopathy was tendon palpation (including pain on palpation, localised tendon thickening or localised swelling). Further objective tests used to assess Achilles tendinopathy included tendon pain during loading activities (single-leg heel raises and hopping) and the Royal London Hospital Test and the Painful Arc Sign. The VISA-A questionnaire as the most commonly used outcome measure to monitor Achilles tendinopathy. However, psychological factors (PES, TKS and PCS) and overall quality of life (SF-12, SF-36 and EQ-5D-5L) were less frequently measured.ConclusionsThere is significant variation in the methodology and outcome measures used to diagnose Achilles tendinopathy. A method for diagnosing Achilles tendinopathy is proposed, that includes both results from the scoping review and recent recommendations for reporting results in tendinopathy. creator: Wesley Matthews creator: Richard Ellis creator: James Furness creator: Wayne A. Hing uri: https://doi.org/10.7717/peerj.12166 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Matthews et al. title: Influence of Pleistocene climatic oscillations on the phylogeography and demographic history of endemic vulnerable trees (section Magnolia) of the Tropical Montane Cloud Forest in Mexico link: https://peerj.com/articles/12181 last-modified: 2021-09-28 description: The Tropical Montane Cloud Forest (TMCF) is a highly dynamic ecosystem that has undergone frequent spatial changes in response to the interglacial-glacial cycles of the Pleistocene. These climatic fluctuations between cold and warm cycles have led to species range shifts and contractions-expansions, resulting in complex patterns of genetic structure and lineage divergence in forest tree species. In this study, we sequenced four regions of the chloroplast DNA (trnT-trnL, trnK5-matk, rpl32-trnL, trnS-trnG) for 20 populations and 96 individuals to evaluate the phylogeography, historical demography, and paleodistributions of vulnerable endemic TMCF trees in Mexico: Magnolia pedrazae (north-region), M. schiedeana (central-region), and M. schiedeana population Oaxaca (south-region). Our data recovered 49 haplotypes that showed a significant phylogeographic structure in three regions: north, central, and south. Bayesian Phylogeographic and Ecological Clustering (BPEC) analysis also supported the divergence in three lineages and highlighted the role of environmental factors (temperature and precipitation) in genetic differentiation. Our historical demography analyses revealed demographic expansions predating the Last Interglacial (LIG, ~125,000 years ago), while Approximate Bayesian Computation (ABC) simulations equally supported two contrasting demographic scenarios. The BPEC and haplotype network analyses suggested that ancestral haplotypes were geographically found in central Veracruz. Our paleodistributions modeling showed evidence of range shifts and expansions-contractions from the LIG to the present, which suggested the complex evolutionary dynamics associated to the climatic oscillations of the Pleistocene. Habitat management of remnant forest fragments where large and genetically diverse populations occur in the three TMCF regions analyzed would be key for the conservation of these magnolia populations. creator: Yessica Rico creator: M. Ángel León-Tapia creator: Marisol Zurita-Solís creator: Flor Rodríguez-Gómez creator: Suria Gisela Vásquez-Morales uri: https://doi.org/10.7717/peerj.12181 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Rico et al. title: Genetic diversity across the mitochondrial genome of eastern oysters (Crassostrea virginica) in the northern Gulf of Mexico link: https://peerj.com/articles/12205 last-modified: 2021-09-28 description: The eastern oyster, Crassostrea virginica, is divided into four populations along the western North Atlantic, however, the only published mitochondrial genome sequence was assembled using one individual in Delaware. This study aimed to (1) assemble C. virginica mitochondrial genomes from Texas with pooled restriction-site-associated DNA sequencing (ezRAD), (2) evaluate the validity of the mitochondrial genome assemblies including comparison with Sanger sequencing data, and (3) evaluate genetic differentiation both between the Delaware and Texas genomes, as well as among three bays in Texas. The pooled-genome-assembled-genomes (PAGs) from Texas exhibited several characteristics indicating that they were valid, including elevated nucleotide diversity in non-coding and the third position of codons, placement as the sister haplotype of the genome from Delaware in a phylogenetic reconstruction of Crassostrea mitochondrial genomes, and a lack of genetic structure in the ND4 gene among the three Texas bays as was found with Sanger amplicons in samples from the same bays several years prior. In the comparison between the Delaware and Texas genome, 27 of 38 coding regions exhibited variability between the two populations, which were differentiated by 273 mutations, versus 1–13 mutations among the Texas samples. Using the full PAGs, there was no additional evidence for population structure among the three Texas bays. While population genetics is rapidly moving towards larger high-density datasets, studies of mitochondrial DNA (and genomes) can be particularly useful for comparing historic data prior to the modern era of genomics. As such, being able to reliably compile mitochondrial genomes from genomic data can improve the ability to compare results across studies. creator: Chani R. Rue creator: Jason D. Selwyn creator: Patricia M. Cockett creator: Bryan Gillis creator: Lauren Gurski creator: Philip Jose creator: Brandi L. Kutil creator: Sharon F. Magnuson creator: Luz Ángela López de Mesa creator: R Deborah Overath creator: Delbert Lee Smee creator: Christopher E. Bird uri: https://doi.org/10.7717/peerj.12205 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Rue et al. title: miR-452-3p inhibited osteoblast differentiation by targeting Smad4 link: https://peerj.com/articles/12228 last-modified: 2021-09-28 description: Osteoblast differentiation is a complex process that is essential for normal bone formation. A growing number of studies have shown that microRNAs (miRNAs) are key regulators in a variety of physiological and pathological processes, including osteogenesis. In this study, BMP2 was used to induce MC3T3-E1 cells to construct osteoblast differentiation cell model. Then, we investigated the effect of miR-452-3p on osteoblast differentiation and the related molecular mechanism by RT-PCR analysis, Western blot analysis, ALP activity, and Alizarin Red Staining. We found that miR-452-3p was significantly downregulated in osteoblast differentiation. Overexpression miR-452-3p (miR-452-3p mimic) significantly inhibited the expression of osteoblast marker genes RUNX2, osteopontin (OPN), and collagen type 1 a1 chain (Col1A1), and decreased the number of calcium nodules and ALP activity. In contrast, knockdown miR-452-3p (miR-452-3p inhibitor) produced the opposite effect. In terms of mechanism, we found that Smad4 may be the target of miR-452-3p, and knockdown Smad4 (si-Smad4) partially inhibited the osteoblast differentiation enhanced by miR-452-3p. Our results suggested that miR-452-3p plays an important role in osteoblast differentiation by targeting Smad4. Therefore, miR-452-3p is expected to be used in the treatment of bone formation and regeneration. creator: Ming Wu creator: Hongyan Wang creator: Dece Kong creator: Jin Shao creator: Chao Song creator: Tieyi Yang creator: Yan Zhang uri: https://doi.org/10.7717/peerj.12228 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Wu et al. title: Application of intra-arterial chemotherapy in high-risk non-muscle invasive bladder cancer: a systematic review and meta-analysis link: https://peerj.com/articles/12248 last-modified: 2021-09-28 description: BackgroundTo summarize the current evidence on the effects of intra-arterial chemotherapy (IAC) on high-risk non-muscle invasive bladder cancer (NMIBC) and compare oncology results with intravesical chemotherapy (IVC).MethodsWe performed a systematic review and cumulative meta-analysis of the primary outcomes of interest by a systematical search of multiple scientific databases in February 2021. The mean difference (MD) and odds ratio (OR) were calculated for continuous and dichotomous variables respectively, with 95% confidence intervals (CIs). The hazard radio (HR) with 95% CIs was used for overall survival (OS), recurrence-free survival (RFS) and progression-free survival (PFS).ResultsA total of six studies with 866 patients were included. For IAC combined with IVC versus IVC alone, statistically significant differences were found regarding tumor recurrence rate (OR: 0.51, 95% CI [0.36∼0.72], p = 0.0001), tumor progression rate (OR: 0.47, 95% CI [0.30∼0.72], p = 0.0006), tumor-specific death rate (OR: 0.49, 95% CI [0.25∼0.99], p = 0.05), PFS (HR: 0.47, 95% CI [0.23∼0.96], p = 0.04) and RFS (HR: 0.60, 95% CI [0.41∼0.87], p = 0.007). No significant difference between two groups was found for time to first recurrence (MD: 3.27, 95% CI [−2.37∼8.92], p = 0.26) and OS (HR: 1.20, 95% CI [0.44∼3.32], p = 0.72). For IAC alone versus IVC, There was no statistical difference in the terms of tumor-specific death rate (OR: 0.67, 95% CI [0.29∼1.53], p = 0.34), RFS (HR: 0.90, 95% CI [0.56∼1.46], p = 0.68) and PFS (HR: 0.71, 95% CI [0.32∼1.55], p = 0.39). Adverse events mainly included nausea/vomiting (36.3%), hypoleukemia (19.4%), neutropenia (16.0%), increased creatinine (9.9%), increased alanine aminotransferase (18.7%), and thrombocytopenia (9.9%).ConclusionThe IAC combined with IVC is a safe and effective treatment for high risk NMIBC, with lower rates of recurrence, progression, tumor-specific death, PFS and RFS, and with minor and tolerable events. The effectiveness of the IAC alone is parallel to the IVC alone. creator: Chengyu You creator: Xianhui Li creator: Yuelin Du creator: Hui Wang creator: Xiaojun Zhang creator: Tangqiang Wei creator: Anguo Wang uri: https://doi.org/10.7717/peerj.12248 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 You et al. title: MINTIA: a metagenomic INserT integrated assembly and annotation tool link: https://peerj.com/articles/11885 last-modified: 2021-09-27 description: The earth harbors trillions of bacterial species adapted to very diverse ecosystems thanks to specific metabolic function acquisition. Most of the genes responsible for these functions belong to uncultured bacteria and are still to be discovered. Functional metagenomics based on activity screening is a classical way to retrieve these genes from microbiomes. This approach is based on the insertion of large metagenomic DNA fragments into a vector and transformation of a host to express heterologous genes. Metagenomic libraries are then screened for activities of interest, and the metagenomic DNA inserts of active clones are extracted to be sequenced and analysed to identify genes that are responsible for the detected activity. Hundreds of metagenomics sequences found using this strategy have already been published in public databases. Here we present the MINTIA software package enabling biologists to easily generate and analyze large metagenomic sequence sets, retrieved after activity-based screening. It filters reads, performs assembly, removes cloning vector, annotates open reading frames and generates user friendly reports as well as files ready for submission to international sequence repositories. The software package can be downloaded from https://github.com/Bios4Biol/MINTIA. creator: Philippe Bardou creator: Sandrine Laguerre creator: Sarah Maman Haddad creator: Sabrina Legoueix Rodriguez creator: Elisabeth Laville creator: Claire Dumon creator: Gabrielle Potocki-Veronese creator: Christophe Klopp uri: https://doi.org/10.7717/peerj.11885 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Bardou et al. title: Efficacy of krill oil versus fish oil on obesity-related parameters and lipid gene expression in rats: randomized controlled study link: https://peerj.com/articles/12009 last-modified: 2021-09-27 description: BackroundThis study aimed to determine the effects of LC n-3 PUFA supplementation on the prevention and treatment of obesity and obesity-related diseases, and to compare the efficiency of different LC n-3 PUFA sources via biochemical and genetic mechanisms in rats.MethodsMale Wistar rats were randomized into four study groups, and fed with a standard diet, High Fat Diet (HFD), HFD+%2.5 Fish Oil (FO-HFD) or HFD+%2.5 Krill Oil (KO-HFD) for eight weeks. Food consumption, weight gain, serum glucose, insulin, ghrelin and leptin concentrations, lipid profile, liver fatty acid composition, and FADS1 and FADS2 mRNA gene expression levels were measured.ResultsWeight gain in each HFD group was significantly higher than control group (p < 0.001), without any differences among them (p < 0.05). LC n-3 PUFAs modified lipid profile, but not glucose tolerance. Serum leptin levels were significantly higher in HFD groups than in the control group, however, no difference in serum ghrelin levels was observed among the groups. Liver n-3 fatty acid desaturation activity was higher (p = 0.74), and liver total lipid content was lower (p = 0.86) in KO-HFD compared to FO-HFD. FADS1 gene expression was highest in the HFD group (p < 0.001) while FADS2 gene expression was highest in the FO-HFD group (p < 0.001).ConclusionLC n-3 PUFAs, especially krill oil, had moderate effects on lipid profile, but limited effects on obesity related parameters, suggesting different effects of different sources on gene expression levels. Further randomized controlled trials are needed to determine the efficacy of different LC n-3 PUFA sources in the prevention and treatment of obesity in humans. creator: Mevra Aydin Cil creator: Atena Ghosi Ghareaghaji creator: Yasin Bayir creator: Zehra Buyuktuncer creator: Halit Tanju Besler uri: https://doi.org/10.7717/peerj.12009 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Aydin Cil et al. title: Evolutionary drivers of the hump-shaped latitudinal gradient of benthic polychaete species richness along the Southeastern Pacific coast link: https://peerj.com/articles/12010 last-modified: 2021-09-27 description: Latitudinal diversity gradients (LDG) and their explanatory factors are among the most challenging topics in macroecology and biogeography. Despite of its apparent generality, a growing body of evidence shows that ‘anomalous’ LDG (i.e., inverse or hump-shaped trends) are common among marine organisms along the Southeastern Pacific (SEP) coast. Here, we evaluate the shape of the LDG of marine benthic polychaetes and its underlying causes using a dataset of 643 species inhabiting the continental shelf (<200 m depth), using latitudinal bands with a spatial resolution of 0.5°, along the SEP (3–56° S). The explanatory value of six oceanographic (Sea Surface Temperature (SST), SST range, salinity, salinity range, primary productivity and shelf area), and one macroecological proxy (median latitudinal range of species) were assessed using a random forest model. The taxonomic structure was used to estimate the degree of niche conservatism of predictor variables and to estimate latitudinal trends in phylogenetic diversity, based on three indices (phylogenetic richness (PDSES), mean pairwise distance (MPDSES), and variation of pairwise distances (VPD)). The LDG exhibits a hump-shaped trend, with a maximum peak of species richness at ca. 42° S, declining towards northern and southern areas of SEP. The latitudinal pattern was also evident in local samples controlled by sampling effort. The random forest model had a high accuracy (pseudo-r2 = 0.95) and showed that the LDG could be explained by four variables (median latitudinal range, SST, salinity, and SST range), yet the functional relationship between species richness and these predictors was variable. A significant degree of phylogenetic conservatism was detected for the median latitudinal range and SST. PDSES increased toward the southern region, whereas VPD showed the opposite trend, both statistically significant. MPDSES has the same trend as PDSES, but it is not significant. Our results reinforce the idea that the south Chile fjord area, particularly the Chiloé region, was likely the evolutionary source of new species of marine polychaetes along SEP, creating a hotspot of diversity. Therefore, in the same way as the canonical LDG shows a decline in diversity while moving away from the tropics; on this case the decline occurs while moving away from Chiloé Island. These results, coupled with a strong phylogenetic signal of the main predictor variables suggest that processes operating mainly at evolutionary timescales govern the LDG. creator: Rodrigo A. Moreno creator: Fabio A. Labra creator: Darko D. Cotoras creator: Patricio A. Camus creator: Dimitri Gutiérrez creator: Luis Aguirre creator: Nicolás Rozbaczylo creator: Elie Poulin creator: Nelson A. Lagos creator: Daniel Zamorano creator: Marcelo M. Rivadeneira uri: https://doi.org/10.7717/peerj.12010 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Moreno et al. title: Long distance (>20 km) downstream detection of endangered stream frogs suggests an important role for eDNA in surveying for remnant amphibian populations link: https://peerj.com/articles/12013 last-modified: 2021-09-27 description: BackgroundGlobally, amphibian species have suffered drastic population declines over the past 40 years. Hundreds of species are now listed as Critically Endangered, with many of these considered “possibly extinct”. Most of these species are stream-dwelling frogs inhabiting remote, montane areas, where remnant populations are hard to find using traditional surveys. Environmental DNA (eDNA) could revolutionize surveys for ‘missing’ and endangered amphibian populations by screening water samples from downstream sections to assess presence in the upstream catchments. However, the utility of this survey technique is dependent on quantifying downstream detection probability and distances.MethodsHere we tested downstream detection distances in two endangered stream frogs (Litoria lorica and L. nannotis) that co-occur in a remote stream catchment in north-east Australia, and for which we know precise downstream distributional limits from traditional surveys. Importantly, the two last populations of L. lorica persist in this catchment: one small (~1,000 frogs) and one very small (~100 frogs). We conducted eDNA screening at a series of sites kilometers downstream from the populations using precipitation from two fixed water volumes (15 and 100 mL) and via water filtering (mean 1,480 L).ResultsWe detected L. nannotis and the small L. lorica population (~1,000 frogs) at most sampling sites, including 22.8 km downstream. The filtration method was highly effective for far-downstream detection, as was precipitation from 100 mL water samples, which also resulted in consistent detections at the far-downstream sites (including to 22.8 km). In contrast, we had limited downstream detection success for the very small L. lorica population (~100 frogs).DiscussionThe ecological aspects of our study system, coupled with thorough traditional surveys, enabled us to measure downstream eDNA detection distances with accuracy. We demonstrate that eDNA from a small population of approximately 1,000 frogs can be detected as far as 22.8 km downstream from the population. Water filtration is considered best for eDNA detection of rare aquatic species—indeed it was effective in this study—but we also achieved far-downstream detections when precipitating eDNA from 100 mL water samples. Collecting small water volumes for subsequent precipitation in the lab is more practical than filtration when surveying remote areas. Our downstream detection distances (>20 km) suggest eDNA is a valuable tool for detecting rare stream amphibians. We provide recommendations on optimal survey methods. creator: Cecilia Villacorta-Rath creator: Conrad J. Hoskin creator: Jan M. Strugnell creator: Damien Burrows uri: https://doi.org/10.7717/peerj.12013 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Villacorta-Rath et al. title: Self-reported body weight and weight-related stigmatization experiences among young adult women—two contexts, but similar attitudes related to body image, mental self-schemas, self-esteem, and stereotypes of people with obesity link: https://peerj.com/articles/12047 last-modified: 2021-09-27 description: BackgroundWeight stigma is a serious challenge because of its negative impact on human health and harmful psychological and behavioral consequences. The aim of the study was to explore and compare the relationships between self-reported body weight and weight-related stigmatization experiences and body image, mental self-image, self-esteem, and stereotypes concerning people with obesity among young adult Polish women (N = 374; aged between 18 and 35).MethodsThe study was conducted online on a Polish sample recruited through a social network site, a website, and snowball sampling. Body mass index (BMI) was used to assign the respondents to groups with normal or excess weight. We tested whether women enrolled in the study experienced weight-related stigmatization using two questions based on the concepts of spoiled identity and related to the obesity stigma. The Contour Drawing Rating Scale was used to study different aspects of the body image and discrepancies between them. The Self-Discrepancy Questionnaire was used to study the self-schemas associated with mental qualities. The Polish version of the Rosenberg’s Self Esteem Scale was applied to determine self-esteem level. Stereotypes concerning people with obesity were studied using the semantic differential method.ResultsAlthough excess weight was associated with weight-related stigmatization experiences, many women reported confronting such stigmatization even though their body weight was normal according to the World Health Organization (WHO) standards. Women with excess weight and women with weight-related stigmatization experiences were characterized by larger discrepancies between the actual body image and the ideal, reflected, and ought body image, lower self-esteem, and more negative beliefs about their mental actual and reflected self compared to women with normal weight and without weight-related stigmatization experiences. The study participants from all groups tended to believe their actual body image to be ampler than the ideal and the ought body images. They also believed that other people perceived their mental qualities more positively than they did. The study groups were also characterized by negative stereotypes of people with obesity, although these stereotypes were more vital in women with excess weight and women who experienced weight-related stigmatization.ConclusionThe study shows the similarity between psychological functioning of women with self-reported excess weight and those who experience weight-related stigma. The results also provide guidelines for practical actions aimed at reducing negative mental outcomes associated with not conforming to body weight standards. creator: Łukasz Jach creator: Sonia Krystoń uri: https://doi.org/10.7717/peerj.12047 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Jach and Krystoń title: Evolution of inbreeding: a gaze into five Italian beef cattle breeds history link: https://peerj.com/articles/12049 last-modified: 2021-09-27 description: In the last decades, intensive selection programs have led to sustained increases of inbreeding in dairy cattle, a feature that might have adverse consequences on the viability and phenotypic performance of their offspring. This study aimed to determine the evolution of inbreeding of five Italian beef cattle breeds (Marchigiana, Chianina, Romagnola, Maremmana, and Podolica) during a period of almost 20 years (2002–2019). The estimates of Ho, He, Fhat2, and Fped averaged across years (2002–2019) in the studied breeds fluctuated between 0.340–0.401, 0.348–0.392, –0.121–0.072, and 0.000–0.068, respectively. Moreover, annual rates of increase of the estimated inbreeding coefficients have been very low (Fhat2 = 0.01–0.02%; Fped = 0.003–0.004%). The use of a high number of bulls combined with strategies implemented by the Association of Italian Beef Cattle Breeders ANABIC to minimize inbreeding might explain these results. Despite the fact that diversity and inbreeding have remained quite stable during the last two decades, we have detected a sustained decrease of the population effective size of these five breeds. Such results should be interpreted with caution due to the inherent difficulty of estimating Ne from SNPs data in a reliable manner. creator: Giacomo Rovelli creator: Maria Gracia Luigi-Sierra creator: Dailu Guan creator: Fiorella Sbarra creator: Andrea Quaglia creator: Francesca Maria Sarti creator: Marcel Amills creator: Emiliano Lasagna uri: https://doi.org/10.7717/peerj.12049 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Rovelli et al. title: Development of droplet digital Polymerase Chain Reaction assays for the detection of long-finned (Anguilla dieffenbachii) and short-finned (Anguilla australis) eels in environmental samples link: https://peerj.com/articles/12157 last-modified: 2021-09-27 description: Freshwater eels are ecologically, and culturally important worldwide. The New Zealand long-finned eel (Anguilla dieffenbachii) and short-finned eel (Anguilla australis) are apex predators, playing an important role in ecosystem functioning of rivers and lakes. Recently, there has been a national decline in their populations due to habitat destruction and commercial harvest. The emergence of targeted environmental DNA detection methodologies provides an opportunity to enhance information about their past and present distributions. In this study we successfully developed species-specific droplet digital Polymerase Chain Reaction (ddPCR) assays to detect A. dieffenbachii and A. australis DNA in water and sediment samples. Assays utilized primers and probes designed for regions of the mitochondrial cytochrome b and 16S ribosomal RNA genes in A. dieffenbachii and A. australis, respectively. River water samples (n = 27) were analyzed using metabarcoding of fish taxa and were compared with the ddPCR assays. The presence of A. dieffenbachii and A. australis DNA was detected in a greater number of water samples using ddPCR in comparison to metabarcoding. There was a strong and positive correlation between gene copies (ddPCR analyses) and relative eel sequence reads (metabarcoding analyses) when compared to eel biomass. These ddPCR assays provide a new method for assessing spatial distributions of A. dieffenbachii and A. australis in a range of environments and sample types. creator: Georgia Thomson-Laing creator: Russleigh Parai creator: Laura T. Kelly creator: Xavier Pochon creator: Rewi Newnham creator: Marcus J. Vandergoes creator: Jamie D. Howarth creator: Susanna A. Wood uri: https://doi.org/10.7717/peerj.12157 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Thomson-Laing et al. title: Evaluation of the molluscicidal activities of arylpyrrole on Oncomelania hupensis, the intermediate host of Schistosoma japonicum link: https://peerj.com/articles/12209 last-modified: 2021-09-27 description: The snail Oncomelania hupensis is the only intermediate host of the highly invasive parasite Schistosoma japonicum. Molluscicide is often used to curb transmission of S. japonicum. Niclosamide, the only World Health Organization (WHO) recognized molluscicide, presents major drawbacks, including high cost and toxicity towards aquatic animals. In the present study, a number of aryl pyrrole derivatives (ADs) were synthesized to serve as potential molluscicides and were tested on O. hupensis. To uncover the underlying mechanisms, adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels were assessed in the soft body of ADs-exposed O. hupensis, using high performance liquid chromatography (HPLC). The effect of C6 on key points of energy metabolism (the activities of complexes I, III, IV and the membrane potential) was determined. We demonstrated that the Compound 6 (C6, 4-bromo-1-(bromomethyl)-2-(4-chlorophenyl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile) exerted the strongest molluscicidal activity against adult O. hupensis at LC50 of 0.27, 0.19, and 0.13 mg/L for 24, 48, and 72 h respectively. Moreover, we found that the bromide on the pyrrole ring of C6 was essential for molluscicidal activity. Furthermore, the ATP content reduced from 194.46 to 139.75 μg/g after exposure to 1/2 LC50, and reduced to 93.06 μg/g after exposure to LC50. ADP, on the other hand, remained the same level before and after C6 exposure. We found that C6, at 1/2 LC50, reduced the membrane potential of O. hupensis, while no significant changes were observed in the activities of complexes I, III, and IV. C6 was identified with excellent activities on O. hupensis. The obtained structure−activity relationship and action mechanism study results should be useful for further compound design and development. creator: Yuntian Xing creator: Jiakai Yao creator: Guoli Qu creator: Jianrong Dai creator: Bainian Feng uri: https://doi.org/10.7717/peerj.12209 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Xing et al. title: Effect of control measures on the pattern of COVID-19 Epidemics in Japan link: https://peerj.com/articles/12215 last-modified: 2021-09-27 description: BackgroundCOVID-19 has spread worldwide since its emergence in 2019. In contrast to many other countries with epidemics, Japan differed in that it avoided lockdowns and instead asked people for self-control. A travel campaign was conducted with a sizable budget, but the number of PCR tests was severely limited. These choices may have influenced the course of the epidemic.MethodsThe increase or decrease in the classes of SARS-CoV-2 variants was estimated by analyzing the published sequences with an objective multivariate analysis. This approach observes the samples in multiple directions, digesting complex differences into simpler forms. The results were compared over time with the number of confirmed cases, PCR tests, and overseas visitors. The kinetics of infection were analyzed using the logarithmic growth rate.ResultsThe declared states of emergency failed to alter the movement of the growth rate. Three epidemic peaks were caused by domestically mutated variants. In other countries, there are few cases in which multiple variants have peaked. However, due to the relaxation of immigration restrictions, several infective variants have been imported from abroad and are currently competing for expansion, creating the fourth peak. By April 2021, these foreign variants exceeded 80%. The chaotic situation in Japan will continue for some time, in part because no effort has been made to identify asymptomatic carriers, and details of the vaccination program are undecided. creator: Tomokazu Konishi uri: https://doi.org/10.7717/peerj.12215 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Konishi title: Heterogeneity in conservation of multifunctional partner enzymes with meiotic importance, CDK2 kinase and BRCA1 ubiquitin ligase link: https://peerj.com/articles/12231 last-modified: 2021-09-27 description: The evolution of proteins can be accompanied by changes not only to their amino acid sequences, but also their structural and spatial molecular organization. Comparison of the protein conservation within different taxonomic groups (multifunctional, or highly specific) allows to clarify their specificity and the direction of evolution. Two multifunctional enzymes, cyclin-dependent kinase 2 (CDK2) and BRCA1 ubiquitin ligase, that are partners in some mitotic and meiotic processes were investigated in the present work. Two research methods, bioinformatics and immunocytochemical, were combined to examine the conservation levels of the two enzymes. It has been established that CDK2 is a highly conserved protein in different taxonomic lineages of the eukaryotic tree. Immunocytochemically, a conserved CDK2 pattern was revealed in the meiotic autosomes of five rodent species and partially in domestic turkey and clawed frog. Nevertheless, variable CDK2 distribution was detected at the unsynapsed segments of the rodent X chromosomes. BRCA1 was shown to be highly conserved only within certain mammalian taxa. It was also noted that in those rodent nuclei, where BRCA1 specifically binds to antigens, asynaptic regions of sex chromosomes were positive. BRCA1 staining was not always accompanied by specific binding, and a high nonspecificity in the nucleoplasm was observed. Thus, the studies revealed different conservation of the two enzymes at the level of protein structure as well as at the level of chromosome behavior. This suggests variable rates of evolution due to both size and configuration of the protein molecules and their multifunctionality. creator: Sergey Matveevsky creator: Tatiana Grishaeva uri: https://doi.org/10.7717/peerj.12231 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Matveevsky and Grishaeva title: The modeled distribution of corals and sponges surrounding the Salas y Gómez and Nazca ridges with implications for high seas conservation link: https://peerj.com/articles/11972 last-modified: 2021-09-24 description: The Salas y Gómez and Nazca ridges are two adjacent seamount chains off the west coast of South America that collectively contain more than 110 seamounts. The ridges support an exceptionally rich diversity of benthic and pelagic communities, with the highest level of endemism found in any marine environment. Despite some historical fishing in the region, the seamounts are relatively pristine and represent an excellent conservation opportunity to protect a global biodiversity hotspot before it is degraded. One obstacle to effective spatial management of the ridges is the scarcity of direct observations in deeper waters throughout the region and an accompanying understanding of the distribution of key taxa. Species distribution models are increasingly used tools to quantify the distributions of species in data-poor environments. Here, we focused on modeling the distribution of demosponges, glass sponges, and stony corals, three foundation taxa that support large assemblages of associated fauna through the creation of complex habitat structures. Models were constructed at a 1 km2 resolution using presence and pseudoabsence data, dissolved oxygen, nitrate, phosphate, silicate, aragonite saturation state, and several measures of seafloor topography. Highly suitable habitat for each taxa was predicted to occur throughout the Salas y Gómez and Nazca ridges, with the most suitable habitat occurring in small patches on large terrain features such as seamounts, guyots, ridges, and escarpments. Determining the spatial distribution of these three taxa is a critical first step towards supporting the improved spatial management of the region. While the total area of highly suitable habitat was small, our results showed that nearly all of the seamounts in this region provide suitable habitats for deep-water corals and sponges and should therefore be protected from exploitation using the best available conservation measures. creator: Samuel Georgian creator: Lance Morgan creator: Daniel Wagner uri: https://doi.org/10.7717/peerj.11972 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Georgian et al. title: New free-living nematode species and records (Chromadorea: Plectida and Desmodorida) from the edge and axis of Kermadec Trench, Southwest Pacific Ocean link: https://peerj.com/articles/12037 last-modified: 2021-09-24 description: One new nematode species is described and two new species records are provided from the edge (6,080 m depth) and axis (7,132 m) of Kermadec Trench, Southwest Pacific. Leptolaimus hadalis sp. nov. is characterised by medium body 587–741 μm long, labial region not offset from body contour, inconspicuous labial sensilla, amphid located 12–19 μm from anterior end, female without supplements, male with four tubular precloacal supplements (alveolar supplements absent), tubular supplements almost straight with dentate tip, arcuate spicules and weakly cuticularized dorsal gubernacular apophyses strongly bent distally. In a previously published ecological survey of Kermadec Trench, L. hadalis sp. nov. was the most abundant species in a core obtained at 8,079 m water depth and third most abundant species in a core obtained at 7,132 m, while only one individual was found at 6,096 m depth, and none at 9,175 m depth (Leduc & Rowden, 2018). Alaimella aff. cincta and Desmodora aff. pilosa are recorded for the first time from the Southwest Pacific region. Prior to the present study, Alaimella had only been recorded from coastal locations and from the Weddell sea to a depth of 2,000 m. The record of Desmodora aff. pilosa at 6,080 m depth is the deepest record of a Desmodora species to date, although unidentified Desmodora specimens have been found as deep as 6,300 m in the South Sandwich Trench. The morphology of the Kermadec Trench Alaimella aff. cincta and Desmodora aff. pilosa specimens bear a strong resemblance to their respective type populations from the Northern Hemisphere, but further morphological and molecular data are required to ascertain whether they in fact represent distinct species. creator: Daniel Leduc uri: https://doi.org/10.7717/peerj.12037 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Leduc title: Quantitative trait locus mapping analysis of multiple traits when using genotype data with potential errors link: https://peerj.com/articles/12187 last-modified: 2021-09-24 description: BackgroundQuantitative trait locus (QTL) analysis aims to locate and estimate the effects of the genes influencing quantitative traits and infer the relationship between gene variants and changes in phenotypic characteristics using statistical methods. Some methods have been developed to map QTLs of multiple traits in the case of no genotype error in a given dataset. However, practical genetic data that people use may contain some potential errors because of the limitations of biotechnology. Common genetic data correction methods can only reduce errors, but cannot calculate the degree of error. In this paper, we propose a QTL mapping strategy for multiple traits in the presence of genotype errors.MethodsThe additive effect, dominant effect, recombination rate, error rate, and other parameters of QTLs can be simultaneously obtained using this new method in the framework of multiple-interval mapping.ResultsOur simulation results show that the accuracy of parameter estimation can be improved by considering the errors of marker genotypes during the analysis of genetic data. Real data analysis also shows that the new method proposed in this paper can map the QTLs of multiple traits more accurately. creator: Liang Tong creator: Ying Zhou creator: Yixing Guo creator: Hui Ding creator: Donghai Ji uri: https://doi.org/10.7717/peerj.12187 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Tong et al. title: Bilateral back squat strength is increased during a 3-week undulating resistance training program with and without variable resistance in DIII collegiate football players link: https://peerj.com/articles/12189 last-modified: 2021-09-24 description: BackgroundOptimizing training adaptations is of the utmost importance for the strength and conditioning professional. The pre-season of any sport is particularly important to ensure preparedness of the athletes. In DIII Collegiate Football pre-season consists of approximately 3 weeks. The abbreviated time of the pre-season increases the importance of optimizing training using safe methods, including alternative loading strategies. The purpose of the current study was to determine if a 3-week variable resistance training VRT during an undulating (UL) resistance training program elicited a greater increase in back squat strength compared to traditional loading methods.Methods and MaterialsForty DIII Football players (age range: 18–25 years) participated in a 3-week UL bilateral back squat (BBS) program. Both groups performed the BBS 3 times per week with a minimum of 24 hours between exercise sessions. The control group (C) (n = 20) (height = 182.3 + 5.1 cm, body mass: pre = 102.8 ± 17.7 kg, post = 104.1 ± 17.8 kg) used traditional loading methods (i.e., Olympic weights only) and the experimental group (E) (n = 20) (height = 180.7 ± 8.0 cm, body mass: pre = 100.3 ± 27.1 kg, post = 101.0 ± 27.7 kg) used traditional loading methods and variable resistance (i.e., resistance bands). The variable resistance accounted for approximately 20% of the total resistance while 80% of the resistance was supplied by traditional loading methods.ResultsWhen all data was pooled, subjects had a significant increase (p < 0.05) in 1-RM BBS from pre (154.2 + 26.1 kg) to post (166.8 + 26.2 kg), with a percent increase of 8.13% at the completion of the 3-week training program. There was no significant difference (p > 0.05) between the C and E groups for muscular strength, muscular power, or vertical jump. Volume-loads were not significantly (p > 0.05) different between groups for any of the weeks (C: Week 1 = 858.1 + 101.3, Week 2 = 588.6 + 69.2, Week 3 = 332.5 + 38.9, Total = 1179.2 + 209.4 vs. E: Week 1 = 835.2 + 179.7, Week 2 = 572.2 + 123.4, Week 3 = 323.5 + 68.8, Total = 1730.9 + 371.8) or for the pre-season as a whole.ConclusionA traditional UL resistance training program and training program with variable resistance are both effective methods at increasing back squat strength during 3 weeks of training. Resistance band variable resistance (VR) does not enhance training effects within a 3-week mesocycle greater than traditional resistance. creator: Jason Sawyer creator: Paul Higgins creator: Paul A. Cacolice creator: Troy Doming uri: https://doi.org/10.7717/peerj.12189 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Sawyer et al. title: Novel real-time PCR based assays for differentiating fall armyworm strains using four single nucleotide polymorphisms link: https://peerj.com/articles/12195 last-modified: 2021-09-24 description: The fall armyworm, Spodoptera frugiperda, is a polyphagous global pest with a preference for gramineous crops such as corn, sorghum and pasture grasses. This species is comprised of two morphologically identical but genetically distinct host strains known as the corn and rice strains, which can complicate pest management approaches. Two molecular markers are commonly used to differentiate between strains, however, discordance between these markers can lead to inconclusive strain identification. Here, we used double digest restriction site associated DNA sequencing to identify diagnostic single nucleotide polymorphisms (SNPs) with alleles unique to each strain. We then used these strain-specific SNPs to develop four real-time PCR based TaqMan assays to rapidly and reliably differentiate between strains and interstrain hybrids. These assays provide a new tool for differentiating between strains in field-collected samples, facilitating future studies on strain population dynamics and interstrain hybridization rates. Understanding the basic ecology of S. frugiperda strains is necessary to inform future management strategies. creator: Ashley E. Tessnow creator: Todd M. Gilligan creator: Eric Burkness creator: Caroline Placidi De Bortoli creator: Juan Luis Jurat-Fuentes creator: Patrick Porter creator: Danielle Sekula creator: Gregory A. Sword uri: https://doi.org/10.7717/peerj.12195 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Tessnow et al. title: Efficacy of 4% articaine vs 2% lidocaine in mandibular and maxillary block and infiltration anaesthesia in patients with irreversible pulpitis: a systematic review and meta-analysis link: https://peerj.com/articles/12214 last-modified: 2021-09-24 description: ObjectiveThe goal of this systematic review and meta-analysis is to determine the performance of 4% Articaine vs. 2% Lidocaine for mandibular and maxillary block and infiltration anaesthesia in patients with irreversible pulpitis (IP).MethodsPubMed/MEDLINE, Cochrane Central Register of Controlled Trials, Web of Science, Google Scholar, and Open Gray were used to conduct a thorough literature search. A manual search of the reference lists of the publications found was also carried out. Two reviewers critically evaluated the papers for inclusion and exclusion criteria, and data extraction was done on the selected publications. The Cochrane Collaboration Tool and the Minors checklist were used to assess the quality of the selected studies for randomised controlled trials (RCTs) and non-randomised studies, respectively. The RevMan software was used to perform a meta-analysis of the pooled data and subgroups according to the technique of anaesthetic solution delivery, as well as a sensitivity analysis (P < 0.05).ResultsA total of twenty-six papers were included in the qualitative synthesis, with twenty-two of them being included in the meta-analysis. There were fifteen studies with a low potential for bias, three with a moderate potential for bias, and seven with a high potential for bias. The combined results of the 19 trials in the tooth level unit revealed that 4% articaine had a success rate 1.37 times greater than 2% lidocaine for mandibular teeth (RR, 1.37; 95% CI [1.17–1.62]; P = 0.0002). For the maxillary buccal infiltration method, the combined results from the three trials revealed that 4% articaine resulted in a success rate 1.06 times greater than 2% lidocaine (RR, 1.06; 95% CI [0.95–1.2]; P = 0.3). Excluding subgroups with a single study in sensitivity analysis for mandibular teeth revealed a substantial improvement in the success rate of the articaine group in treating IP when compared to the lidocaine group.ConclusionThe findings of this meta-analysis back up the claim that articaine is more effective than lidocaine in providing anaesthesia in patients with IP. PROSPERO Registration No.: CRD42020204606 (https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020204606). creator: Sanjay Miglani creator: Irfan Ansari creator: Swadheena Patro creator: Ankita Mohanty creator: Shahnaz Mansoori creator: Bhoomika Ahuja creator: Mohmed Isaqali Karobari creator: Krishna Prasad Shetty creator: Musab Hamed Saeed creator: Alexander Maniangat Luke creator: Ajinkya M. Pawar uri: https://doi.org/10.7717/peerj.12214 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Miglani et al. title: Locating ligand binding sites in G-protein coupled receptors using combined information from docking and sequence conservation link: https://peerj.com/articles/12219 last-modified: 2021-09-24 description: GPCRs (G-protein coupled receptors) are the largest family of drug targets and share a conserved structure. Binding sites are unknown for many important GPCR ligands due to the difficulties of GPCR recombinant expression, biochemistry, and crystallography. We describe our approach, ConDockSite, for predicting ligand binding sites in class A GPCRs using combined information from surface conservation and docking, starting from crystal structures or homology models. We demonstrate the effectiveness of ConDockSite on crystallized class A GPCRs such as the beta2 adrenergic and A2A adenosine receptors. We also demonstrate that ConDockSite successfully predicts ligand binding sites from high-quality homology models. Finally, we apply ConDockSite to predict the ligand binding sites on a structurally uncharacterized GPCR, GPER, the G-protein coupled estrogen receptor. Most of the sites predicted by ConDockSite match those found in other independent modeling studies. ConDockSite predicts that four ligands bind to a common location on GPER at a site deep in the receptor cleft. Incorporating sequence conservation information in ConDockSite overcomes errors introduced from physics-based scoring functions and homology modeling. creator: Ashley Ryan Vidad creator: Stephen Macaspac creator: Ho Leung Ng uri: https://doi.org/10.7717/peerj.12219 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Vidad et al. title: Vitamin D serum levels and non-melanoma skin cancer risk link: https://peerj.com/articles/12234 last-modified: 2021-09-24 description: BackgroundSkin cancer is one of the common malignancies. There is sufficient evidence that sunlight (ultraviolet radiation) contributes to the development of skin cancer, but there is also evidence that relates adequate serum levels of vitamin D produced on the skin by the action of ultraviolet radiation with the decreased risk of various types of cancers, including skin cancer. The aim of this study was to investigate the association of vitamin D serum levels among patients with non-melanoma skin cancers (basal cell carcinoma and squamous cell carcinoma) and controls.MethodsA prospective observational case-control study was conducted in a sample of 84 subjects in Extremadura (Spain). Forty-one patients with histologically diagnosed basal cell carcinomas and squamous cell carcinomas and 43 healthy controls were randomly chosen to assess whether vitamin D (25(OH)D3) serum level, age and sex were related to non-melanoma skin cancer and to determine the possible risk of this type of skin cancer for these variables.ResultsWhen analysing serum vitamin D levels, we ensured that all our subjects, both cases and controls, had normal or low serum vitamin D levels, even though the samples were taken during months with the highest solar irradiance in our region. It is striking in our results that there was a higher percentage of subjects with deficits of vitamin D who did not have skin cancer (66%) than patients with deficits with these types of skin cancers (34%). When adjusting the model for age and sex, vitamin D values above 18 ng/ml increased the risk of suffering from non-melanoma skin cancer by nearly 7-fold (aOR: 6.94, 95% CI [1.55–31.11], p = 0.01).ConclusionsDespite the controversial data obtained in the literature, our results suggest that lower levels of vitamin D may be related to a reduced incidence of non-melanoma skin cancer. creator: Carolina Morgado-Águila creator: Guadalupe Gil-Fernández creator: Orlando Rafael Dávila-Villalobos creator: Jesús Pérez-Rey creator: Purificación Rey-Sánchez creator: Francisco José Rodríguez-Velasco uri: https://doi.org/10.7717/peerj.12234 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Morgado-Águila et al. title: Pristimantis achupalla sp. n., a new minute species of direct-developing frog (Amphibia, Anura, Strabomantidae) inhabiting bromeliads of the montane forest of the Amazonian Andes of Puno, Peru link: https://peerj.com/articles/11878 last-modified: 2021-09-23 description: We describe a new species of bromeliad-dwelling Pristimantis from primary montane forest (2,225 m a.s.l.) in southern Peru. The type locality is near Thiuni, in the Department of Puno (province of Carabaya) in the upper watershed of a tributary of the Inambari River. Pristimantis achupalla sp. n. is characterized by a snout-vent length of 10.0–12.8 mm in adult males (n = 4), unknown in adult females, and is compared morphologically and genetically with species in the Pristimantis lacrimosus group, and with other similar species of Pristimantis. The new species is characterized by having skin on dorsum and flanks rugose, green brownish color, distinctive scapular folds, subacuminate or acuminate snout profile, upper eyelid bearing two or three subconical tubercles and some rounded tubercles, rostral papilla, flanks light brown to brown, with irregular dark brown marks. creator: Alex Ttito creator: Alessandro Catenazzi uri: https://doi.org/10.7717/peerj.11878 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Ttito and Catenazzi title: A predictive model for the ichnological suitability of the Jezero crater, Mars: searching for fossilized traces of life-substrate interactions in the 2020 Rover Mission Landing Site link: https://peerj.com/articles/11784 last-modified: 2021-09-23 description: Ichnofossils, the fossilized products of life-substrate interactions, are among the most abundant biosignatures on Earth and therefore they may provide scientific evidence of potential life that may have existed on Mars. Ichnofossils offer unique advantages in the search for extraterrestrial life, including the fact that they are resilient to processes that obliterate other evidence for past life, such as body fossils, as well as chemical and isotopic biosignatures. The goal of this paper is evaluating the suitability of the Mars 2020 Landing Site for ichnofossils. To this goal, we apply palaeontological predictive modelling, a technique used to forecast the location of fossil sites in uninvestigated areas on Earth. Accordingly, a geographic information system (GIS) of the landing site is developed. Each layer of the GIS maps the suitability for one or more ichnofossil types (bioturbation, bioerosion, biostratification structures) based on an assessment of a single attribute (suitability factor) of the Martian environment. Suitability criteria have been selected among the environmental attributes that control ichnofossil abundance and preservation in 18 reference sites on Earth. The goal of this research is delivered through three predictive maps showing which areas of the Mars 2020 Landing Site are more likely to preserve potential ichnofossils. On the basis of these maps, an ichnological strategy for the Perseverance rover is identified, indicating (1) 10 sites on Mars with high suitability for bioturbation, bioerosion and biostratification ichnofossils, (2) the ichnofossil types, if any, that are more likely to be present at each site, (3) the most efficient observation strategy for detecting eventual ichnofossils. The predictive maps and the ichnological strategy can be easily integrated in the existing plans for the exploration of the Jezero crater, realizing benefits in life-search efficiency and cost-reduction. creator: Andrea Baucon creator: Carlos Neto de Carvalho creator: Antonino Briguglio creator: Michele Piazza creator: Fabrizio Felletti uri: https://doi.org/10.7717/peerj.11784 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Baucon et al. title: Dietary effects on fitness in captive-reared Hawaiian tree snails link: https://peerj.com/articles/11789 last-modified: 2021-09-23 description: The native terrestrial snail fauna of the Hawaiian Islands faces numerous threats that have led to severe range reductions, population declines, and extinction of species. With the continued declines of many wild populations, a crucial component of preserving Hawaiian terrestrial snail biodiversity is through captive rearing programs, like that implemented by the Hawai‘i Department of Land and Natural Resources Snail Extinction Prevention Program. Rare and endangered tree snails in the family Achatinellidae, which feed on epiphytic microbial communities, are maintained in captivity with a diet that includes native vegetation brought in from nearby forests, as well as a cultured fungus originally isolated from native host trees. Recent mortality events in lab populations have been attributed to wild-gathered vegetation. These events have increased interest in developing a completely manufactured or cultured diet that would eliminate the need for exposure to wild-gathered plants. This study compared survival and egg production in Auriculella diaphana provided with lab-cultured fungus, and those provided with wild vegetation. We compared the number of eggs laid and number of deaths among three treatments: (1) wild collected vegetation only; (2) wild vegetation supplemented with laboratory-cultured fungus; and (3) laboratory cultured fungus only. Mortality did not significantly differ among treatments, but the number of eggs laid was significantly higher in snails provided wild vegetation and cultured fungus (F = 24.998; P < 0.001), compared with those provided with only wild vegetation (t = 1.88, P = 0.032) or only cultured fungus (t = 4.530, P = 0.004). Our results suggest: (1) the existing strain of cultured fungus alone is not sufficient to maintain captive-reared snail populations; (2) the additional energy or calcium provided by the cultured fungus appears to enhance egg reproduction in captive-reared populations; (3) the presence or absence of live vegetation influences snail behavior, including aestivation and egg laying. These results highlight the importance of ongoing research to culture additional species of fungi at a rate that could support captive-reared populations, as the diversity of fungi present in wild epiphytic microbial communities may be important for snail reproductive health. creator: Evan Strouse creator: Melissa R. Price creator: David R. Sischo uri: https://doi.org/10.7717/peerj.11789 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Strouse et al. title: A four-gene prognostic signature for predicting the overall survival of patients with lung adenocarcinoma link: https://peerj.com/articles/11911 last-modified: 2021-09-23 description: BackgroundThe prognosis of patients for lung adenocarcinoma (LUAD) is known to vary widely; the 5-year overall survival rate is just 63% even for the pathological IA stage. Thus, in order to identify high-risk patients and facilitate clinical decision making, it is vital that we identify new prognostic markers that can be used alongside TNM staging to facilitate risk stratification.MethodsWe used mRNA expression from The Cancer Genome Atlas (TCGA) cohort to identify a prognostic gene signature and combined this with clinical data to develop a predictive model for the prognosis of patients for lung adenocarcinoma. Kaplan-Meier curves, Lasso regression, and Cox regression, were used to identify specific prognostic genes. The model was assessed via the area under the receiver operating characteristic curve (AUC-ROC) and validated in an independent dataset (GSE50081) from the Gene Expression Omnibus (GEO).ResultsOur analyses identified a four-gene prognostic signature (CENPH, MYLIP, PITX3, and TRAF3IP3) that was associated with the overall survival of patients with T1-4N0-2M0 in the TCGA dataset. Multivariate regression suggested that the total risk score for the four genes represented an independent prognostic factor for the TCGA and GEO cohorts; the hazard ratio (HR) (high risk group vs low risk group) were 2.34 (p < 0.001) and 2.10 (p = 0.017). Immune infiltration estimations, as determined by an online tool (TIMER2.0) showed that CD4+ T cells were in relative abundance in the high risk group compared to the low risk group in both of the two cohorts (both p < 0.001). We established a composite prognostic model for predicting OS, combined with risk-grouping and clinical factors. The AUCs for 1-, 3-, 5- year OS in the training set were 0.750, 0.737, and 0.719; and were 0.645, 0.766, and 0.725 in the validation set. The calibration curves showed a good match between the predicted probabilities and the actual probabilities.ConclusionsWe identified a four-gene predictive signature which represents an independent prognostic factor and can be used to identify high-risk patients from different TNM stages of LUAD. A new prognostic model that combines a prognostic gene signature with clinical features exhibited better discriminatory ability for OS than traditional TNM staging. creator: Lei Liu creator: Huayu He creator: Yue Peng creator: Zhenlin Yang creator: Shugeng Gao uri: https://doi.org/10.7717/peerj.11911 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Liu et al. title: Organic farming practices change the soil bacteria community, improving soil quality and maize crop yields link: https://peerj.com/articles/11985 last-modified: 2021-09-23 description: BackgroundThe importance of organic farming has increased through the years to promote food security allied with minimal harm to the ecosystem. Besides the environmental benefits, a recurring problem associated with organic management is the unsatisfactory yield. A possible solution may rely on the soil microbiome, which presents a crucial role in the soil system. Here, we aimed to evaluate the soil bacterial community structure and composition under organic and conventional farming, considering the tropical climate and tropical soil.MethodologyOur organic management treatments were composed by composted poultry manure and green manure with Bokashi. Both organic treatments were based on low nitrogen inputs. We evaluated the soil bacterial community composition by high-throughput sequencing of 16S rRNA genes, soil fertility, and soil enzyme activity in two organic farming systems, one conventional and the last transitional from conventional to organic.ResultsWe observed that both organic systems evaluated in this study, have higher yield than the conventional treatment, even in a year with drought conditions. These yield results are highly correlated with changes in soil chemical properties and enzymatic activity. The attributes pH, Ca, P, alkaline phosphatase, and β- glucosidase activity are positively correlated with organic systems, while K and Al are correlated with conventional treatment. Also, our results show in the organic systems the changes in the soil bacteria community, being phyla Acidobacteria, Firmicutes, Nitrospirae, and Rokubacteria the most abundant. These phyla were correlated with soil biochemical changes in the organic systems, helping to increase crop yields.ConclusionDifferent organic management systems, (the so-called natural and organic management systems, which use distinct organic sources), shift the soil bacterial community composition, implying changes in their functionalities. Also, our results contributed to the identification of target bacterial groups and changes in soil chemical properties and enzymatic activity in a trophic organic farming system, which may contribute to higher crop yields. creator: Ademir Durrer creator: Thiago Gumiere creator: Maurício Rumenos Guidetti Zagatto creator: Henrique Petry Feiler creator: Antonio Marcos Miranda Silva creator: Rodrigo Henriques Longaresi creator: Sérgio K. Homma creator: Elke J.B.N. Cardoso uri: https://doi.org/10.7717/peerj.11985 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Durrer et al. title: Near-natural transformation of Pinus tabuliformis better improve soil nutrients and soil microbial community link: https://peerj.com/articles/12098 last-modified: 2021-09-23 description: Pinus tabulaeformis plantations have been established around northern China to restore degraded land and provide timber or fuelwood. In recent years, widely distributed monoculture P. tabulaeformis forests have been transformed into mixed forests due to various ecological problems. However, the current research on the influence of near-natural transformation of P. tabulaeformis on soil microbial diversity and community composition remains limited. Therefore, we examined the effect of forest conversion from monoculture Pinus tabuliformis (PT) to P. tabuliformis-Armeniaca vulgaris (PTAU), P. tabuliformis - Robinia pseudoacacia (PTRP), P. tabuliformis - Vitex negundo L. var. heterophylla (PTVN) forests on soil microbial community diversity and composition. The results indicated that compared to PT, PTAU, PTVN, and PTRP could enhance the soil pH, TC, TN, AN, and AK in different degrees, the most obvious in PTAU. Near-natural transformation of P. tabuliformis could improve soil bacterial Pielou_e index, and Simpson index, as well as soil fungal Chao1 index. Proteobacteria and Ascomycota were the dominant soil microbial community at the phylum level. What’s more, both soil bacterial and fungal community among PT, PTAU, PTRP and PTVN showed clear different, and PTAU obviously altered the soil microbial community structure. Proteobacteria was the predominant group in PT, while, Gemmatimonadetes enriched in PTVN. Ascomycota was the predominant group in PTAU, while, Basidiomycota was the predominant group in PTRP. Near-natural transformation of P. tabuliformis could change soil microbial community via altering soil characteristics. In brief, our research results revealed the influence of tree composition and soil nutrient availability on soil microbial diversity and composition, and provided management guidance for introduction soil microbial community in forest protection and management. creator: You Yin creator: Qiuli Li creator: Haitao Du uri: https://doi.org/10.7717/peerj.12098 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Yin et al. title: Evaluation of exotic oat (Avena sativa L.) varieties for forage and grain yield in response to different levels of nitrogen and phosphorous link: https://peerj.com/articles/12112 last-modified: 2021-09-23 description: A field experiment was conducted during the Rabi season 2017–2018 (October–March) at the University of Agriculture, Peshawar research farm to examine the influence of different nitrogen (N) and phosphorus (P) levels on two different oat varieties: Australian and Ukrainian. The treatments included control and three levels of nitrogen and phosphorus at 30, 60, and 90 kg ha−1. The treatments were arranged in randomized complete block design (RCBD) and replicated three times. The findings showed that the oat varieties were significantly different from one another in yield and yield parameters. The Australian variety recorded higher emergence (49 plants m−2), days to emergence (15 days), days to flowering (122 days), days to maturity (145 days), plant height (142.7 cm), number of leaves (6.03 leaves plant−1), number of tillers (92.2 tillers m−1), biological yield (8,179.2 kg ha−1), and grain yield (3,725.6 kg ha−1) than the Ukrainian variety. Similarly, different N and P levels, the maximum days to emergence, days to flowering, and days to maturity were recorded in a control plot. The application of 105 kg N + 90 kg P ha−1 was statistically similar to the application of 105 kg N + 60 kg P ha−1. Maximum emergence (60 plants m−2), number of leaves (7.0 leaves plant−1), plant height (118.6 cm), number of tillers m−1 (102.6), biological yield (9,687.5 kg ha−1), and grain yield (4,416.7 kg ha−1) were determined in Australian variety. Based on the findings of this study, the Australian variety performed better in terms of yield and yield components and the application of N and P fertilizers at the rate of 105 kg N + 60 kg P ha−1 produced the best results in both oat varieties. creator: Hamida Bibi creator: Suleman Hameed creator: Mudassar Iqbal creator: Amal Al-Barty creator: Hadeer Darwish creator: Amanullah Khan creator: Shazma Anwar creator: Ishaq Ahmad Mian creator: Murad Ali creator: Afia Zia creator: Muhammad Irfan creator: Maria Mussarat uri: https://doi.org/10.7717/peerj.12112 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Bibi et al. title: Land use changes in Zhangjiakou from 2005 to 2025 and the importance of ecosystem services link: https://peerj.com/articles/12122 last-modified: 2021-09-23 description: Changes in local land use affect regional ecological services, development planning, and optimal use of space. We analyzed the effects of changes in land use from 2000 to 2025 on the spatial distribution of ecosystem services using CLUS-S modeling to evaluate ecosystem functions in Zhangjiakou, China. We found that the urban ecosystem area in Zhangjiakou increased and farmland decreased between 2000–2025. Water conservation was relatively high and was concentrated in the nature reserves of southern Zhangjiakou. Soil conservation was mainly distributed in eastern and southern counties. The results of the CLUE-S model showed that the relative operating characteristics of the six land use types were > 0.70, and the logistic regression equation was able to successfully explain the distribution pattern of the different types of land use. creator: Kaipeng Xu creator: Yanyan Chi creator: Rongfeng Ge creator: Xiahui Wang creator: Siyang Liu uri: https://doi.org/10.7717/peerj.12122 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Xu et al. title: C3 plant isotopic variability in a boreal mixed woodland: implications for bison and other herbivores link: https://peerj.com/articles/12167 last-modified: 2021-09-23 description: Plant isotopic baselines are critical for accurately reconstructing ancient diets and environments and for using stable isotopes to monitor ecosystem conservation. This study examines the stable carbon and nitrogen isotope compositions (δ13C, δ15N) of terrestrial C3 plants in Elk Island National Park (EINP), Alberta, Canada, with a focus on plants consumed by grazers. EINP is located in a boreal mixed woodland ecozone close to the transition area between historic wood and plains bison habitats, and is currently home to separate herds of wood and plains bison. For this study, 165 C3 plant samples (grasses, sedges, forbs, shrubs, and horsetail) were collected from three habitat types (open, closed, and wet) during two seasons (summer and fall). There were no statistically significant differences in the δ13C or δ15N values of grasses, sedges, shrubs and forbs. On the other hand, plant δ13C and δ15N values varied among habitats and plant parts, and the values increased from summer to fall. These results have several implications for interpreting herbivore tissue isotopic compositions: (1) consuming different proportions of grasses, sedges, shrubs, and forbs might not result in isotopic niche partitioning, (2) feeding in different microhabitats or selecting different parts of the same types of plants could result in isotopic niche partitioning, and (3) seasonal isotopic changes in herbivore tissues could reflect seasonal isotopic changes in dietary plants rather than (or in addition to) changes in animal diet or physiology. In addition, the positively skewed plant δ15N distributions highlight the need for researchers to carefully evaluate the characteristics of their distributions prior to reporting data (e.g., means, standard deviations) or applying statistical models (e.g., parametric tests that assume normality). Overall, this study reiterates the importance of accessing ecosystem-specific isotopic baselines for addressing research questions in archaeology, paleontology, and ecology. creator: Jessica Z. Metcalfe uri: https://doi.org/10.7717/peerj.12167 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Metcalfe title: Comparative analysis of codon usage patterns in chloroplast genomes of five Miscanthus species and related species link: https://peerj.com/articles/12173 last-modified: 2021-09-23 description: Miscanthus is not only a perennial fiber biomass crop, but also valuable breeding resource for its low-nutrient requirements, photosynthetic efficiency and strong adaptability to environment. In the present study, the codon usage patterns of five different Miscanthus plants and other two related species were systematically analyzed. The results indicated that the cp genomes of the seven representative species were preference to A/T bases and A/T-ending codons. In addition, 21 common high-frequency codons and 4–11 optimal codons were detected in the seven chloroplast genomes. The results of ENc-plot, PR2-plot and neutrality analysis revealed the codon usage patterns of the seven chloroplast genomes are influenced by multiple factors, in which nature selection is the main influencing factor. Comparative analysis of the codon usage frequencies between the seven representative species and four model organisms suggested that Arabidopsis thaliana, Populus trichocarpa and Saccharomyces cerevisiae could be considered as preferential appropriate exogenous expression receptors. These results might not only provide important reference information for evolutionary analysis, but also shed light on the way to improve the expression efficiency of exogenous gene in transgenic research based on codon optimization. creator: Jiajing Sheng creator: Xuan She creator: Xiaoyu Liu creator: Jia Wang creator: Zhongli Hu uri: https://doi.org/10.7717/peerj.12173 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Sheng et al. title: Effect of vascular resection for perihilar cholangiocarcinoma: a systematic review and meta-analysis link: https://peerj.com/articles/12184 last-modified: 2021-09-23 description: ObjectiveTo evaluate the effect of vascular resection (VR), including portal vein resection (PVR) and hepatic artery resection (HAR), on short- and long-term outcomes in patients with perihilar cholangiocarcinoma (PHC).BackgroundResection surgery and transplantation are the main treatment methods for PHC that provide a chance of long-term survival. However, the efficacy and safety of VR, including PVR and HAR, for treating PHC remain controversial.MethodsThis study was registered at the International Prospective Register of Systematic Reviews (CRD42020223330). The EMBASE, PubMed, and Cochrane Library databases were used to search for eligible studies published through November 28, 2020. Studies comparing short- and long-term outcomes between patients who underwent hepatectomy with or without PVR and/or HAR were included. Random- and fixed-effects models were applied to assess the outcomes, including morbidity, mortality, and R0 resection rate, as well as the impact of PVR and HAR on long-term survival.ResultsTwenty-two studies including 4,091 patients were deemed eligible and included in this study. The meta-analysis showed that PVR did not increase the postoperative morbidity rate (odds ratio (OR): 1.03, 95% confidenceinterval (CI): [0.74–1.42], P = 0.88) and slightly increased the postoperative mortality rate (OR: 1.61, 95% CI [1.02–2.54], P = 0.04). HAR did not increase the postoperative morbidity rate (OR: 1.32, 95% CI [0.83–2.11], P = 0.24) and significantly increased the postoperative mortality rate (OR: 4.20, 95% CI [1.88–9.39], P = 0.0005). Neither PVR nor HAR improved the R0 resection rate (OR: 0.70, 95% CI [0.47–1.03], P = 0.07; OR: 0.77, 95% CI [0.37–1.61], P = 0.49, respectively) or long-term survival (OR: 0.52, 95% CI [0.35–0.76], P = 0.0008; OR: 0.43, 95% CI [0.32–0.57], P < 0.00001, respectively).ConclusionsPVR is relatively safe and might benefit certain patients with advanced PHC in terms of long-term survival, but it is not routinely recommended. HAR results in a higher mortality rate and lower overall survival rate, with no proven benefit. creator: Yong Liu creator: Guangbing Li creator: Ziwen Lu creator: Tao Wang creator: Yang Yang creator: Xiaoyu Wang creator: Jun Liu uri: https://doi.org/10.7717/peerj.12184 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Liu et al. title: Adam21 is dispensable for reproductive processes in mice link: https://peerj.com/articles/12210 last-modified: 2021-09-23 description: BackgroundAs a group of membrane-anchored proteins, the proteins containing a disintegrin and metalloprotease domain (ADAMs) control many biological processes, especially for male fertility. Mouse Adam21 was previously found to be specifically expressed in the somatic cells and germ cells of testes, but its functional role during spermatogenesis and male reproductive processes is still unknown.MethodsAdam21-null mice were created using the CRISPR/Cas9 system. Quantitative real-time PCR was used for analyzing of gene expression. Histological, cytological and immunofluorescence staining were performed to analyze the phenotypes of mouse testis and epididymis. Intracellular lipid droplets (LDs) were detected by Oil red O (ORO) staining and BODIPY staining. Fertility and sperm characteristics were also detected.ResultsHere, we successfully generated an Adam21 conventional knockout mouse model via CRISPR/Cas9 technology so that we can explore its potential role in male reproduction. We found that male mice lacking Adam21 have normal fertility without any detectable defects in spermatogenesis or sperm motility. Histological analysis of the seminiferous epithelium showed no obvious spermatogenesis difference between Adam21-null and wild-type mice. Cytological analysis revealed no detectable defects in meiotic progression, neither Sertoli cells nor Leydig cells displayed any defect compared with that of the control mice. All these results suggest that Adam21 might not be essential for male fertility in mice, and its potential function still needs further investigation. creator: Yinghong Chen creator: Chao Liu creator: Yongliang Shang creator: Liying Wang creator: Wei Li creator: Guoping Li uri: https://doi.org/10.7717/peerj.12210 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Chen et al. title: Ontogenetic moulting behavior of the Cambrian oryctocephalid trilobite Arthricocephalites xinzhaiheensis link: https://peerj.com/articles/12217 last-modified: 2021-09-23 description: Moulting behaviors in trilobites are a crucial strategy during development. Previous studies have demonstrated inter-and intraspecific variability of moulting behavior in trilobites. Currently, ecdysial motifs for trilobites are considered not stable even within species and fewer detailed studies dealt with moulting behaviors in a single species of trilobite during development. Here a large sample of meraspid to holaspid exuviae of Arthricocephalites xinzhaiheensis (131 specimens) from the Cambrian Balang Formation of South China has allowed description of the reasonably complete ontogenic moulting sequence. Both ontogenetic stage and body size reveal gradual transition of configuration from Somersault configuration to Henningsmoen’s configuration during development. Somersault configuration is exclusive till meraspid degree five and exists in subsequent growth stages. This suggests that opening of the facial and rostral sutures allowing the emergence forward of the post-ecdysial trilobite was prevalent in early growth stages. In later development, Henningsmoen’s configuration (showing disarticulation of the cranidium) became more dominant. This study indicates that gradual transition of ontogenetic moulting behavior occurred in oryctocephalid trilobites in the early Cambrian. creator: Yifan Wang creator: Jin Peng creator: Dezhi Wang creator: Hui Zhang creator: Xiuchun Luo creator: Yunbin Shao creator: Quanyi Sun creator: Chenchen Ling creator: Qiujun Wang uri: https://doi.org/10.7717/peerj.12217 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Wang et al. title: Large-scale differences in diversity and functional adaptations of prokaryotic communities from conserved and anthropogenically impacted mangrove sediments in a tropical estuary link: https://peerj.com/articles/12229 last-modified: 2021-09-23 description: Mangroves are tropical ecosystems with strategic importance for climate change mitigation on local and global scales. They are also under considerable threat due to fragmentation degradation and urbanization. However, a complete understanding of how anthropogenic actions can affect microbial biodiversity and functional adaptations is still lacking. In this study, we carried out 16S rRNA gene sequencing analysis using sediment samples from two distinct mangrove areas located within the Serinhaém Estuary, Brazil. The first sampling area was located around the urban area of Ituberá, impacted by domestic sewage and urban runoff, while the second was an environmentally conserved site. Our results show significant changes in the structure of the communities between impacted and conserved sites. Biodiversity, along with functional potentials for the cycling of carbon, nitrogen, phosphorus and sulfur, were significantly increased in the urban area. We found that the environmental factors of organic matter, temperature and copper were significantly correlated with the observed shifts in the communities. Contributions of specific taxa to the functional potentials were negatively correlated with biodiversity, such that fewer numbers of taxa in the conserved area contributed to the majority of the metabolic potential. The results suggest that the contamination by urban runoff may have generated a different environment that led to the extinction of some taxa observed at the conserved site. In their place we found that the impacted site is enriched in prokaryotic families that are known human and animal pathogens, a clear negative effect of the urbanization process. creator: Carolina O. De Santana creator: Pieter Spealman creator: Vania Melo creator: David Gresham creator: Taise de Jesus creator: Eddy Oliveira creator: Fabio Alexandre Chinalia uri: https://doi.org/10.7717/peerj.12229 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 De Santana et al. title: Reliability of the weight-bearing ankle dorsiflexion range of motion measurement using a smartphone goniometer application link: https://peerj.com/articles/11977 last-modified: 2021-09-22 description: BackgroundWeight-bearing ankle dorsiflexion range of motion measurement (weight-bearing lunge test) is gaining in popularity because it mimics lower extremity function in daily physical activities. The purpose of the study is to assess the intra-rater and the inter-rater reliability of the weight-bearing ankle dorsiflexion range of motion measurement with a flexed knee using a smartphone application Spirit Level Plus installed on an Android smartphone.MethodsThirty-two young, healthy subjects participated in the study and were measured in four sessions by two examiners. One measurement was taken on each ankle in every session. Eight measurements were taken from each participant. A total of 256 were taken from all the participants. The measurements for the individual subject were repeated no sooner than 24 hours after the first session. In order to assess the reliability, intraclass correlation coefficients (ICC), standard error measurements (SEM) and minimal detectable change (MDC) at the 95% confidence interval were calculated.ResultsStatistical data analysis revealed moderate intra-rater reliability for the right ankle (ICC = 0.72, 95% CI [0.49–0.85]) and good intra-rater reliability for the left ankle (ICC = 0.82, 95% CI [0.66–0.91]). Inter-rater reliability is moderate for the right (ICC = 0.73, 95% CI [0.52–0.86]) and the left ankle (ICC = 0.65, 95% CI [0.39–0.81]).ConclusionThe observed method is moderately reliable and appropriate when the main objective is to assess ankle dorsiflexion mobility in weight-bearing when weight-bearing is not contraindicated. The concurrent validity of the Spirit Level Plus application is excellent. creator: Helena Zunko creator: Renata Vauhnik uri: https://doi.org/10.7717/peerj.11977 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Zunko and Vauhnik title: Transcriptome and structure analysis in root of Casuarina equisetifolia under NaCl treatment link: https://peerj.com/articles/12133 last-modified: 2021-09-22 description: BackgroundHigh soil salinity seriously affects plant growth and development. Excessive salt ions mainly cause damage by inducing osmotic stress, ion toxicity, and oxidation stress. Casuarina equisetifolia is a highly salt-tolerant plant, commonly grown as wind belts in coastal areas with sandy soils. However, little is known about its physiology and the molecular mechanism of its response to salt stress.ResultsEight-week-old C. equisetifolia seedlings grown from rooted cuttings were exposed to salt stress for varying durations (0, 1, 6, 24, and 168 h under 200 mM NaCl) and their ion contents, cellular structure, and transcriptomes were analyzed. Potassium concentration decreased slowly between 1 h and 24 h after initiation of salt treatment, while the content of potassium was significantly lower after 168 h of salt treatment. Root epidermal cells were shed and a more compact layer of cells formed as the treatment duration increased. Salt stress led to deformation of cells and damage to mitochondria in the epidermis and endodermis, whereas stele cells suffered less damage. Transcriptome analysis identified 10,378 differentially expressed genes (DEGs), with more genes showing differential expression after 24 h and 168 h of exposure than after shorter durations of exposure to salinity. Signal transduction and ion transport genes such as HKT and CHX were enriched among DEGs in the early stages (1 h or 6 h) of salt stress, while expression of genes involved in programmed cell death was significantly upregulated at 168 h, corresponding to changes in ion contents and cell structure of roots. Oxidative stress and detoxification genes were also expressed differentially and were enriched among DEGs at different stages.ConclusionsThese results not only elucidate the mechanism and the molecular pathway governing salt tolerance, but also serve as a basis for identifying gene function related to salt stress in C. equisetifolia. creator: Yujiao Wang creator: Jin Zhang creator: Zhenfei Qiu creator: Bingshan Zeng creator: Yong Zhang creator: Xiaoping Wang creator: Jun Chen creator: Chonglu Zhong creator: Rufang Deng creator: Chunjie Fan uri: https://doi.org/10.7717/peerj.12133 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Wang et al. title: Detection of syrup adulterants in manuka and jarrah honey using HPTLC-multivariate data analysis link: https://peerj.com/articles/12186 last-modified: 2021-09-22 description: High-Performance Thin-Layer Chromatography (HPTLC) was used in a chemometric investigation of the derived sugar and organic extract profiles of two different honeys (Manuka and Jarrah) with adulterants. Each honey was adulterated with one of six different sugar syrups (rice, corn, golden, treacle, glucose and maple syrups) in five different concentrations (10%, 20%, 30%, 40%, and 50% w/w). The chemometric analysis was based on the combined sugar and organic extract profiles’ datasets. To obtain the respective sugar profiles, the amount of fructose, glucose, maltose, and sucrose present in the honey was quantified and for the organic extract profile, the honey’s dichloromethane extract was investigated at 254 and 366 nm, as well as at T (Transmittance) white light and at 366 nm after derivatisation. The presence of sugar syrups, even at a concentration of only 10%, significantly influenced the honeys’ sugar and organic extract profiles and multivariate data analysis of these profiles, in particular cluster analysis (CA), principal component analysis (PCA), principal component regression (PCR), partial least-squares regression (PLSR) and Machine Learning using an artificial neural network (ANN), were able to detect post-harvest syrup adulterations and to discriminate between neat and adulterated honey samples. Cluster analysis and principal component analysis, for instance, could easily differentiate between neat and adulterated honeys through the use of CA or PCA plots. In particular the presence of excess amounts of maltose and sucrose allowed for the detection of sugar adulterants and adulterated honeys by HPTLC-multivariate data analysis. Partial least-squares regression and artificial neural networking were employed, with augmented datasets, to develop optimal calibration for the adulterated honeys and to predict those accurately, which suggests a good predictive capacity of the developed model. creator: Md Khairul Islam creator: Kevin Vinsen creator: Tomislav Sostaric creator: Lee Yong Lim creator: Cornelia Locher uri: https://doi.org/10.7717/peerj.12186 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Islam et al. title: Microbial growth in biobeds for treatment of residual pesticide in banana plantations link: https://peerj.com/articles/12200 last-modified: 2021-09-22 description: BackgroundHigh doses of ethylenebisdithiocarbamate (EBDC) are used in banana production, and unused pesticide mixture (solution) is often disposed of improperly. This can result in soil and water contamination and present an undue risk to rural communities and the environment. An alternative to reduce the environmental impacts caused by pesticide residues is the biobeds treatment. It is necessary to establish if the composition of the proposed biomixtures supports microbial activity to degrade pesticides in biobeds. This research aimed to evaluate the EBDC effect on the distribution and abundance of microbial populations in polluted biomixtures .MethodsFor this purpose, a biomixture based on banana stem, mulch, and Fluvisol soil (50:25:25% v/v) was prepared and polluted with 1,000 mg L−1 EBDC. The response variables kinetics were determined every 14 days for three months, such as pH, organic matter, moisture, cation exchange capacity, microbial colonies, and cell counts at three depths within the experimental units.ResultsEBDC reduced the number of microbial colonies by 72%. Bacterial cells rapidly decreased by 69% and fungi 89% on the surface, while the decrease was gradual and steady at the middle and bottom of the biobed. The microbial populations stabilized at day 42, and the bacteria showed a total recovery on day 84, but the fungi slightly less. At the end of the experiment, the concentration of EBDC in the biomixture was 1.3–4.1 mg L−1. A correlation was found between fungal count (colonies and cells) with EBDC concentration. A replacement of the biomixture is suggested if the bacterial population becomes less than 40 × 106 CFU mL−1 and the fungal population less than 8 × 104 CFU mL−1 or if the direct cell count becomes lower than 50 × 104 cells mL−1 in bacteria and 8 × 102 cells mL−1 in fungi.ConclusionThe biomixture based on banana stem supports the microbial activity necessary for the degradation of the EBDC pesticide. It was found that fungi could be used as indicators of the pollutant degradation process in the biomixtures. Microbial counts were useful to establish the mobility and degradation time of the pesticide and the effectiveness of the biomixture. Based on the results, it is appropriate to include the quantification of microbial populations to assess the effectiveness of pesticide degradation and the maturity level of the biomixture. creator: Verónica I. Domínguez-Rodríguez creator: Eduardo Baltierra-Trejo creator: Rodolfo Gómez-Cruz creator: Randy H. Adams uri: https://doi.org/10.7717/peerj.12200 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Domínguez-Rodríguez et al. title: Chimpanzee facial gestures and the implications for the evolution of language link: https://peerj.com/articles/12237 last-modified: 2021-09-22 description: Great ape manual gestures are described as communicative, flexible, intentional, and goal-oriented. These gestures are thought to be an evolutionary pre-cursor to human language. Conversely, facial expressions are thought to be inflexible, automatic, and derived from emotion. However, great apes can make a wide range of movements with their faces, and they may possess the control needed to gesture with their faces as well as their hands. We examined whether chimpanzee facial expressions possess the four important gesture properties and how they compare to manual gestures. To do this, we quantified variables that have been previously described through largely qualitative means. Chimpanzee facial expressions met all four gesture criteria and performed remarkably similar to manual gestures. Facial gestures have implications for the evolution of language. If other mammals also show facial gestures, then the gestural origins of language may be much older than the human/great ape lineage. creator: Brittany Florkiewicz creator: Matthew Campbell uri: https://doi.org/10.7717/peerj.12237 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Florkiewicz and Campbell title: Fungi in soil and understory have coupled distribution patterns link: https://peerj.com/articles/11915 last-modified: 2021-09-21 description: Ecological processes that control fungal distribution are not well understood because many fungi can persist in a wide variety of dissimilar habitats which are seldom sampled simultaneously. Geographic range size is reflective of species’ resource usage, and for plants and animals, there is a robust positive correlation between niche-breadth and range-size. It remains unknown whether this pattern is true for fungi. To investigate the fungal niche breadth–range size relationship we identified habitat specialists and generalists from two habitats (plant leaves and soil) and asked whether habitat specialization influenced fungal biogeography. We sampled fungi from the soil and phylloplane of tropical forests in Vanuatu and used DNA metabarcoding of the fungal ITS1 region to examine rarity, range size, and habitat connectivity. Fungal communities from the soil and phylloplane are spatially autocorrelated and the spatial distribution of individual fungal OTU are coupled between habitats. Habitat breadth (generalist fungi) did not result in larger range sizes but did correlate positively with occurrence frequency. Fungi that were frequently found were also found in high abundance, a common observation in similar studies of plants and animals. Fungal abundance-occupancy relationships differed by habitat and habitat-specificity. Soil specialists were found to be locally abundant but restricted geographically. In contrast, phylloplane generalists were found to be abundant over a large range in multiple habitats. These results are discussed in the context of differences between habitat characteristics, stability and spatial distribution. Identifying factors that drive spatial variation is key to understanding the mechanisms that maintain biodiversity in forests. creator: André Boraks creator: Anthony S. Amend uri: https://doi.org/10.7717/peerj.11915 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Boraks and Amend title: Who’s who in Magelona: phylogenetic hypotheses under Magelonidae Cunningham & Ramage, 1888 (Annelida: Polychaeta) link: https://peerj.com/articles/11993 last-modified: 2021-09-21 description: Known as shovel head worms, members of Magelonidae comprise a group of polychaetes readily recognised by the uniquely shaped, dorso-ventrally flattened prostomium and paired ventro-laterally inserted papillated palps. The present study is the first published account of inferences of phylogenetic hypotheses within Magelonidae. Members of 72 species of Magelona and two species of Octomagelona were included, with outgroups including members of one species of Chaetopteridae and four of Spionidae. The phylogenetic inferences were performed to causally account for 176 characters distributed among 79 subjects, and produced 2,417,600 cladograms, each with 404 steps. A formal definition of Magelonidae is provided, represented by a composite phylogenetic hypothesis explaining seven synapomorphies: shovel-shaped prostomium, prostomial ridges, absence of nuchal organs, ventral insertion of palps and their papillation, presence of a burrowing organ, and unique body regionation. Octomagelona is synonymised with Magelona due to the latter being paraphyletic relative to the former. The consequence is that Magelonidae is monotypic, such that Magelona cannot be formally defined as associated with any phylogenetic hypotheses. As such, the latter name is an empirically empty placeholder, but because of the binomial name requirement mandated by the International Code of Zoological Nomenclature, the definition is identical to that of Magelonidae. Several key features for future descriptions are suggested: prostomial dimensions, presence/absence of prostomial horns, morphology of anterior lamellae, presence/absence of specialised chaetae, and lateral abdominal pouches. Additionally, great care must be taken to fully describe and illustrate all thoracic chaetigers in descriptions. creator: Kate Mortimer creator: Kirk Fitzhugh creator: Ana Claudia dos Brasil creator: Paulo Lana uri: https://doi.org/10.7717/peerj.11993 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Mortimer et al. title: The syndemic condition of psychosocial problems related to depression among sexually transmitted infections patients link: https://peerj.com/articles/12022 last-modified: 2021-09-21 description: BackgroundThe prevalence of depression in sexually transmitted infections (STIs) patients is much higher than general public. However, studies focusing on comprehensive psychosocial effects on depression among STIs patients are limited. This study aimed to examine association of multiple psychosocial syndemic conditions with depression among STIs patients in Shanghai, China.MethodsWe conducted a cross-sectional study and recruited 910 STIs patients from Shanghai Skin Disease Hospital. Participants self-reported their demographics and themselves completed the scales of depression, self-esteem, loneliness, social support, entrapment, defeat and interpersonal needs. Logistic regressions were performed to detect the possible contributing psychosocial factors for depression and to verify the syndemic conditions of psychosocial problems.ResultsOf the STIs patient sample, the prevalence of depression was 17.9%. Multivariable analysis showed low-level self-esteem (odds ratio [ORm]: 2.18, 95% CI [1.19–4.00]) and social support (ORm: 2.18, 95% CI [1.37–3.46]), high-level entrapment (ORm: 6.31, 95% CI [3.75–10.62]) and defeat (ORm: 2.60, 95% CI [1.51–4.48]) increased the risk of depression. Psychosocial syndemic conditions magnified effect in fusing depression (adjusted odds ratio [AOR]: 11.94, 95% CI [7.70–18.53]). Participants with more than 4 psychosocial problems were about 22 times more likely to have depression (AOR: 22.12, 95% CI [13.19–37.09]).ConclusionsThe psychosocial problems syndemic magnifying the risk of depression was confirmed and psychosocial interventions to prevent depression is needed among STIs patients. creator: Chen Xu creator: Yuan Shi creator: Xiaoyue Yu creator: Ruijie Chang creator: Huwen Wang creator: Hui Chen creator: Rongxi Wang creator: Yujie Liu creator: Shangbin Liu creator: Yong Cai creator: Yang Ni creator: Suping Wang uri: https://doi.org/10.7717/peerj.12022 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Xu et al. title: LitR directly upregulates autoinducer synthesis and luminescence in Aliivibrio logei link: https://peerj.com/articles/12030 last-modified: 2021-09-21 description: LitR is a master-regulator of transcription in the ainS/R and luxS/PQ quorum sensing (QS) systems of bacteria from Vibrio and Aliivibrio genera. Here, we for the first time directly investigated the influence of LitR on gene expression in the luxI/R QS system of psychrophilic bacteria Aliivibrio logei. Investigated promoters were fused with Photorhabdus luminescens luxCDABE reporter genes cassette in a heterological system of Escherichia coli cells, litR A. logei was introduced into the cells under control of Plac promoter. LitR has been shown to upregulate genes of autoinducer synthase (luxI), luciferase and reductase (luxCDABE), and this effect doesn’t depend on presence of luxR gene. To a much lesser degree, LitR induces luxR1, but not the luxR2 — the main luxI/R regulator. Enhanced litR expression leads to an increase in a LuxI-autoinducer synthesis and a subsequent LuxR-mediated activation of the luxI/R QS system. Effect of LitR on luxI transcription depends on lux-box sequence in luxI promoter even in absence of luxR (lux-box is binding site of LuxR). The last finding indicates a direct interaction of LitR with the promoter in the lux-box region. Investigation of the effect of LitR A. logei on luxI/R QS systems of mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio salmonicida showed direct luxR-independent upregulation of luxI and luxCDABE genes. To a lesser degree, it induces luxR A. fischeri and luxR1 A. salmonicida. Therefore, we assume that the main role of LitR in cross-interaction of these three QS systems is stimulating the expression of luxI. creator: Sergey Bazhenov creator: Olga Melkina creator: Vadim Fomin creator: Ekaterina Scheglova creator: Pavel Krasnik creator: Svetlana Khrulnova creator: Gennadii Zavilgelsky creator: Ilya Manukhov uri: https://doi.org/10.7717/peerj.12030 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Bazhenov et al. title: LncRNAs specifically overexpressed in endocervical adenocarcinoma are associated with an unfavorable recurrence prognosis and the immune response link: https://peerj.com/articles/12116 last-modified: 2021-09-21 description: BackgroundCervical cancer is the fourth most common gynecological tumor in terms of both the incidence and mortality of females worldwide. Cervical squamous cell carcinoma (CSCC) accounts for 70–80% of cervical cancers, and endocervical adenocarcinoma (EAC) accounts for 20–25%. Unlike CSCC, EAC has worse clinical outcomes and prognosis. In this study, we explored the relationship between various types of long noncoding RNAs (lncRNAs) and pathological types of cervical cancer.MethodsRNA sequencing (RNA-Seq) and clinical data from The Cancer Genome Atlas (TCGA) were used in this study. A single-sample gene set enrichment analysis (ssGSEA) and the ESTIMATE package were used to assess lncRNA activity and immune responses, respectively. RT-qPCR was performed to verify our findings.ResultsWe explored the relationship between various types of lncRNAs and pathological types of cervical cancer. A series of long intergenic noncoding RNAs (lincRNAs) and antisense RNAs, which are the major types of lncRNAs, were identified to be specifically expressed in EAC and associated with a poor recurrence prognosis in patients with cervical cancer, suggesting that they might serve as independent prognostic markers of recurrence in patients with cervical cancer. RT-qPCR was performed to verify the 10 EAC-specific lncRNAs in cervical cancer samples we collected. Furthermore, the overexpression of these lncRNAs was positively correlated with EAC pathology levels but negatively correlated with immune responses in the microenvironment of cervical cancer.ConclusionsThese lncRNAs potentially represent new biomarkers for the prediction of the recurrence prognosis and help obtain deeper insights into potential immunotherapeutic approaches for treating cervical cancer. creator: Yong Song creator: Long Nie creator: Yu-Ting Zhang uri: https://doi.org/10.7717/peerj.12116 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Song et al. title: Mass mortality of pearl oyster (Pinctada fucata (Gould)) in Japan in 2019 and 2020 is caused by an unidentified infectious agent link: https://peerj.com/articles/12180 last-modified: 2021-09-21 description: Mass mortality of 0-year-old pearl oysters, Pinctada fucata (Gould), and anomalies in adults were observed in Japan’s major pearl farming areas in the summer of 2019 and 2020. Although adult oyster mortality was low, both adult and juvenile oysters underwent atrophy of the soft body, detachment of the mantle from nacre (the shiny inner surface of the valves), deposition of brownish material on the nacre, and loss of nacre luster. Infection trials were conducted to verify the involvement of pathogens in this phenomenon. Healthy adult pearl oysters were obtained from areas where this disease had not occurred to use as the recipients. The sources of infection were either affected adult oysters with atrophied soft bodies or batches of juveniles in which mortality had reached conspicuous levels. Transmission of the disease to the healthy oysters were tested either by cohabitation with affected oysters or by injections of the hemolymph of affected animals. The injection infection test examined the effects of filtration and chloroform exposure on the pathogen. Occurrence of the disease was confirmed by the appearance of brown deposits on the nacre and loss of nacre luster. The abnormalities of nacre were clearly reproduced in recipient shells in three out of four cohabitation trials with affected oysters. The disease was also reproduced in six out of six injection trails either with hemolymph filtered through 100 nm filter or with hemolymph treated with chloroform. In a serial passage with hemolymph injections, the disease was successfully transmitted through eight passages. These results suggest that the etiology of the disease is a non-enveloped virus with a diameter ≤100 nm. creator: Tomomasa Matsuyama creator: Satoshi Miwa creator: Tohru Mekata creator: Yuta Matsuura creator: Tomokazu Takano creator: Chihaya Nakayasu uri: https://doi.org/10.7717/peerj.12180 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Matsuyama et al. title: Graft survival of Pinus engelmannii Carr. in relation to two grafting techniques with dormant and sprouting buds link: https://peerj.com/articles/12182 last-modified: 2021-09-21 description: Developing methods for successfully grafting forest species will be helpful for establishing asexual seed orchards and increasing the success of forest genetic improvement programs in Mexico. In this study we investigated the effects of two grafting techniques (side veneer and top cleft) and two phenological stages of the scion buds (end of latency and beginning of sprouting), in combination with other seven grafting variables, on the sprouting and survival of 120 intraspecific grafts of Pinus engelmannii Carr. The scions used for grafting were taken from a 5.5-year-old commercial forest plantation. The first grafting was performed on January 18 (buds at the end of dormancy) and the second on February 21 (buds at the beginning of sprouting). The data were examined by analysis of variance and a test of means and were fitted to two survival models (the Weibull’s accelerated failure time and the Cox’s proportional hazards model) and the respective hazard ratios were calculated. Survival was higher in the top cleft grafts made with buds at the end of latency, with 80% sprouting and an estimated average survival time of between 164 and 457 days after the end of the 6-month evaluation period. Four variables (grafting technique, phenological stage of the scion buds, scion diameter and rootstock height) significantly affected the risk of graft death in both survival models. Use of top cleft grafts with buds at the end of the latency stage, combined with scion diameters smaller than 11.4 mm and rootstock heights greater than 58.5 cm, was associated with a lower risk of death. creator: Alberto Pérez-Luna creator: José Ciro Hernández-Díaz creator: Christian Wehenkel creator: Sergio Leonel Simental-Rodríguez creator: Javier Hernández-Velasco creator: José Ángel Prieto-Ruíz uri: https://doi.org/10.7717/peerj.12182 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Pérez-Luna et al. title: Impact of urbanization on functional diversity in macromycete communities along an urban ecosystem in Southwest Mexico link: https://peerj.com/articles/12191 last-modified: 2021-09-21 description: Macromycetes are a group of fungi characterized by the production of fruit bodies and are highly relevant in most terrestrial ecosystems as pathogens, mutualists, and organic matter decomposers. Habitat transformation can drastically alter macromycete communities and diminish the contribution of these organisms to ecosystem functioning; however, knowledge on the effect of urbanization on macrofungal communities is scarce. Diversity metrics based on functional traits of macromycete species have shown to be valuable tools to predict how species contribute to ecosystem functionality since traits determine the performance of species in ecosystems. The aim of this study was to assess patterns of species richness, functional diversity, and composition of macrofungi in an urban ecosystem in Southwest Mexico, and to identify microclimatic, environmental, and urban factors related to these patterns in order to infer the effect of urbanization on macromycete communities. We selected four oak forests along an urbanization gradient and established a permanent sampling area of 0.1 ha at each site. Macromycete sampling was carried out every week from June to October 2017. The indices used to measure functional diversity were functional richness (FRic), functional divergence (FDig), and functional evenness (FEve). The metric used to assess variation of macrofungal ecological function along the study area was the functional value. We recorded a total of 134 macromycete species and 223 individuals. Our results indicated a decline of species richness with increased urbanization level related mainly to microclimatic variables, and a high turnover of species composition among study sites, which appears to be related to microclimatic and urbanization variables. FRic decreased with urbanization level, indicating that some of the available resources in the niche space within the most urbanized sites are not being utilized. FDig increased with urbanization, which suggests a high degree of niche differentiation among macromycete species within communities in urbanized areas. FEve did not show notable differences along the urbanization gradient, indicating few variations in the distribution of abundances within the occupied sections of the niche space. Similarly, the functional value was markedly higher in the less urbanized site, suggesting greater performance of functional guilds in that area. Our findings suggest that urbanization has led to a loss of macromycete species and a decrease in functional diversity, causing some sections of the niche space to be hardly occupied and available resources to be under-utilized, which could, to a certain extent, affect ecosystem functioning and stability. creator: Marko Gómez-Hernández creator: Emily Avendaño-Villegas creator: María Toledo-Garibaldi creator: Etelvina Gándara uri: https://doi.org/10.7717/peerj.12191 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Gómez-Hernández et al. title: Multiomics-based analyses of KPNA2 highlight its multiple potentials in hepatocellular carcinoma link: https://peerj.com/articles/12197 last-modified: 2021-09-21 description: Dysregulation and prognostic roles of Karyopherin α2 (KPNA2) were reported in many malignancies including hepatocellular carcinoma (HCC). A multi-omics analysis of KPNA2 is needed to gain a deeper understanding of its multilevel molecular characteristics and provide novel clues for HCC diagnosis, prognosis, and target therapy. Herein multi-omic alterations of KPNA2 were analyzed at genetic, epigenetic, transcript, and protein levels with evaluation of their relevance with clinicopathological features of HCC by integrative analyses. The significant correlations of KPNA2 expression with its gene copy number variation (CNV) and methylation status were shown through Spearman correlation analyses. With Cox regression, Kaplan-Meier survival, and receiver operating characteristic (ROC) analyses, based on the factors of KPNA2 CNV, methylation, expression, and tumor stage, risk models for HCC overall survival (OS) and disease-free survival (DFS) were constructed which could discriminate the 1-year, 3-year, and 5-year OS/DFS status effectively. With Microenvironment Cell Populations-counter (MCP-counter), the immune infiltrations of HCC samples were evaluated and their associations with KPNA2 were shown. KPNA2 expression in liver was found to be influenced by low fat diet and presented significant correlations with fatty acid metabolism and fatty acid synthase activity in HCC. KPNA2 was detected lowered in HCC patient’s plasma by enzyme linked immunosorbent assay (ELISA), consistent with its translocation to nuclei of HCC cells. In conclusion, KPNA2 multilevel dysregulation in HCC and its correlations with immune infiltration and the fatty acid metabolism pathway indicated its multiple roles in HCC. The clinicopathological significance of KPNA2 was highlighted through the in-depth analyses at multilevels. creator: Jinzhong Zhang creator: Xiuzhi Zhang creator: Lingxiao Wang creator: Chunyan Kang creator: Ningning Li creator: Zhefeng Xiao creator: Liping Dai uri: https://doi.org/10.7717/peerj.12197 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Zhang et al. title: Key auxin response factor (ARF) genes constraining wheat tillering of mutant dmc link: https://peerj.com/articles/12221 last-modified: 2021-09-21 description: Tillering ability is a key agronomy trait for wheat (Triticum aestivum L.) production. Studies on a dwarf monoculm wheat mutant (dmc) showed that ARF11 played an important role in tillering of wheat. In this study, a total of 67 ARF family members were identified and clustered to two main classes with four subgroups based on their protein structures. The promoter regions of T. aestivum ARF (TaARF) genes contain a large number of cis-acting elements closely related to plant growth and development, and hormone response. The segmental duplication events occurred commonly and played a major role in the expansion of TaARFs. The gene collinearity degrees of the ARFs between wheat and other grasses, rice and maize, were significantly high. The evolution distances among TaARFs determine their expression profiles, such as homoeologous genes have similar expression profiles, like TaARF4-3A-1, TaARF4-3A-2 and their homoeologous genes. The expression profiles of TaARFs in various tissues or organs indicated TaARF3, TaARF4, TaARF9 and TaARF22 and their homoeologous genes played basic roles during wheat development. TaARF4, TaARF9, TaARF12, TaARF15, TaARF17, TaARF21, TaARF25 and their homoeologous genes probably played basic roles in tiller development. qRT-PCR analyses of 20 representative TaARF genes revealed that the abnormal expressions of TaARF11 and TaARF14 were major causes constraining the tillering of dmc. Indole-3-acetic acid (IAA) contents in dmc were significantly less than that in Guomai 301 at key tillering stages. Exogenous IAA application significantly promoted wheat tillering, and affected the transcriptions of TaARFs. These data suggested that TaARFs as well as IAA signaling were involved in controlling wheat tillering. This study provided valuable clues for functional characterization of ARFs in wheat. creator: Junchang Li creator: Yumei Jiang creator: Jing Zhang creator: Yongjing Ni creator: Zhixin Jiao creator: Huijuan Li creator: Ting Wang creator: Peipei Zhang creator: Wenlong Guo creator: Lei Li creator: Hongjie Liu creator: Hairong Zhang creator: Qiaoyun Li creator: Jishan Niu uri: https://doi.org/10.7717/peerj.12221 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Li et al. title: Response of plant diversity and soil physicochemical properties to different gap sizes in a Pinus massoniana plantation link: https://peerj.com/articles/12222 last-modified: 2021-09-21 description: As one means of close-to-nature management, forest gaps have an important impact on the ecological service function of plantations. To improve the current situation of P. massoniana plantations, three different sizes of forest gaps (large gaps, medium gaps and small gaps) were established to observe whether gap setting can improve the soil fertility and plant diversity of forest plantations. The results showed that compared with the control, the soil organic matter content of different soil layers increased significantly in the medium forest gap and large forest gap. The content of soil organic matter in the surface layer of the middle gap had the largest increase (80.64%). Compared with the control, the content of soil-available potassium between different soil layers decreased significantly by 15.93% to 25.80%. The soil hydrolysable nitrogen reached its maximum under the medium gap. Soil moisture showed significant changes among different gap treatments, different soil layers and their interaction, decreasing significantly in large gaps and small gaps but increasing significantly in medium gaps. The soil bulk density decreased significantly compared with the control, and the surface soil reached the minimum in the medium gap. There were different plant species in forest gaps of different sizes, and shrub layer plants were more sensitive to gap size differences than herb layer plants. The plant diversity indices of the shrub layer increased significantly and showed a maximum under the medium gap. The plant diversity of the herb layer showed the opposite trend, and the Shannon-Wiener index, Simpson index and Pielou index were significantly lower than those of the control. RDA showed that different gap treatments had significant effects on the distribution of plants under the forest. Soil available potassium, soil moisture and soil bulk density affected the distribution and diversity of plants under the forest, serving as the limiting factors of plant growth. In forest management, if we strictly consider the improvement of plant diversity and soil physicochemical properties, these results suggest that a medium gap should be established in a plantation for natural restoration. creator: Qian Lyu creator: Jiangli Liu creator: Junjie Liu creator: Yan Luo creator: Luman Chen creator: Gang Chen creator: Kuangji Zhao creator: Yuqin Chen creator: Chuan Fan creator: Xianwei Li uri: https://doi.org/10.7717/peerj.12222 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Lyu et al. title: Cortinarius subsalor and C. tibeticisalor spp. nov., two new species from the section Delibuti from China link: https://peerj.com/articles/11982 last-modified: 2021-09-20 description: Cortinarius subsalor and C. tibeticisalor, belonging to the section Delibuti, are described from China as new to science. Cortinarius subsalor has been found to be associated with Lithocarpus trees in subtropical China and resembling C. salor, but it differs from the later by having slender basidiomata and the narrower basidiospores. Cortinarius tibeticisalor was collected from eastern Tibetan Plateau, associated with Abies. It differs from other species within sect. Delibuti by having olive tinge of mature or dried basidiomata and bigger basidiospores. The molecular data also support C. subsalor and C. tibeticisalor as new species. The phylogenetic analyses and biogeography of sect. Delibuti are discussed and a key to the species of this section currently known in the world is provided. creator: Meng-Le Xie creator: Jun-Liang Chen creator: Chayanard Phukhamsakda creator: Bálint Dima creator: Yong-Ping Fu creator: Rui-Qing Ji creator: Ke Wang creator: Tie-Zheng Wei creator: Yu Li uri: https://doi.org/10.7717/peerj.11982 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Xie et al. title: The association between serum CD4 T lymphocyte counts and surgical outcomes in HIV/AIDS patients in Guangxi, China: a retrospective cohort study link: https://peerj.com/articles/12023 last-modified: 2021-09-20 description: BackgroundHIV/AIDS is a chronic disease leading to complications in infected individuals that often require surgical intervention. These patients’ serum CD4 T lymphocyte (CD4) counts represent one of the most important indicators of their ability to tolerate surgical treatment. Previous studies have demonstrated that CD4 cell count (CD4-CC) < 200 cells/μl may increase the risk of surgical complications in these patients, limiting their ability to undergo surgery, which may negatively affect their quality of life. Further investigation into the surgical outcomes of patients with CD4-CC < 200 cells/μl should provide guidance in making appropriate clinical decisions for the optimal healthcare of this patient demographic.MethodsAll enrolled patients were selected from 14 prefecture-level general hospitals in Guangxi, China, and were referred to AIDS outpost hospitals for inpatient surgical therapy. A total cohort of 168 adult patients was retrospectively analyzed. Multifactorial and stratified analyses were performed to evaluate the in surgical outcome differences for patients with CD4-CC < 200 cells/μl (N = 43), using those with CD4-CC ≥ 200 cells/μl (N = 125) as controls.ResultsPoor incisional healing was used as the primary outcome indicator, and postoperative complications were used as the secondary outcome indicator. In the patient group with CD4-CC < 200 cells/μl, the risk of surgical complications was significantly increased (OR 2.379; 95% CI [1.049–5.394]) after adjustment. Adjusted stratified analysis of the CD4-CC < 200 cells/μl group revealed that individuals over 60 years (OR 27.504; 95% CI [2.297–329.317]) with erythrocyte counts below 4.00/ml for males or 3.50/ml for females (OR 3.353; 95% CI [1.079–10.419]) had a significantly higher risk of postoperative complications; this finding was statistically different from the control (CD4 ≥ 200 cells/μl) group. However, there was no significant difference between the two groups regarding the risk of poorly healed incision outcomes.ConclusionsPreliminary findings suggest that a serum CD4-CC < 200 cells/μl is not a definitive contraindication for surgical therapy and that baseline and surgical characteristics may help predict surgical outcomes in these patients. Further studies are needed to confirm these findings. creator: Aimei Liu creator: Cunxu Liu creator: Xiaojun Deng creator: Yongbao Huang creator: Linchu Liao creator: Zhihao Meng creator: Minfu He creator: Junli Huang uri: https://doi.org/10.7717/peerj.12023 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Liu et al. title: Diverse traits of aquatic plants cannot individually explain their consumption by the generalist gastropod Biomphalairia glabrata link: https://peerj.com/articles/12031 last-modified: 2021-09-20 description: Several experimental studies on aquatic plants have reported the prevalence of chemical defense mechanism against herbivory, as opposed to structural, life-forms or other traits. Here, our laboratory feeding experiments and integrative analysis explored the relationship among palatability (fresh or reconstituted plants used as artificial diet) and various chemical/nutritional traits (i.e., contents of dry mass, ash, nitrogen, protein, and phenols) of diverse aquatic plants and their susceptibility to consumption by the generalist gastropod Biomphalaria glabrata. Biomphalaria glabrata consumed all of the assayed aquatic plants in a hierarchical yet generalized way, with the consumption of fresh plants, their reconstituted forms and defensive properties of lipophilic extracts not being significantly correlated with plant physical or chemical traits to determine the feeding preference of the gastropod. Our results do not reveal a prevalence for a specific plant attribute contributing to herbivory. Instead, they indicate that the susceptibility of aquatic plants to generalist consumers is probably related to a combination of their chemical and physical properties, resulting in moderate grazing rates by generalist consumers. creator: Renato Crespo Pereira creator: Nathália Nocchi creator: Tatiana U.P. Konno creator: Angelica R. Soares uri: https://doi.org/10.7717/peerj.12031 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Pereira et al. title: A comprehensive review and call for studies on firefly larvae link: https://peerj.com/articles/12121 last-modified: 2021-09-20 description: BackgroundFireflies (Coleoptera: Lampyridae) are commonly recognized by adult traits, such as a soft exoskeleton, lanterns and associated glow and flash patterns, but their larval stage is far less appreciated. However, fireflies spend most of their lives as larvae, and adults of most species rely solely on resources previously obtained. Therefore, studying the immature stages is imperative towards a comprehensive understanding of fireflies. This paper reviews and indicates key gaps in the biology of firefly larvae based on available literature.MethodologyWe reviewed the literature on firefly larvae to identify key issues and important taxonomic, geographic, and subject biases and gaps.ResultsWe found 376 papers that included information on firefly larvae. Only 139 species in 47 genera across eight of eleven lampyrid subfamilies have been studied during larval stages. These numbers reveal a staggering gap, since 94% of species and over half of the genera of fireflies were never studied in a crucial stage of their life cycle. Most studies on firefly larvae focus on two subfamilies (Luciolinae and Lampyrinae) in four zoogeographic regions (Sino-Japanese, Oriental, Nearctic, and Palearctic), whereas the other subfamilies and regions remain largely unstudied. These studies mainly dealt with morphology and behavior, other subjects remaining greatly understudied by comparison, including habitats, life cycle, physiology and interactions.ConclusionsTogether, these literature biases and gaps highlight how little is known about firefly larvae, and warmly invite basic and applied research, in the field and in the lab, to overcome these limitations and improve our understanding of firefly biology to better preserve them. creator: William B. Riley creator: Simone Policena Rosa creator: Luiz Felipe Lima da Silveira uri: https://doi.org/10.7717/peerj.12121 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Riley et al. title: SARS-CoV-2 seroprevalence in a high-altitude setting in Peru: adult population-based cross-sectional study link: https://peerj.com/articles/12149 last-modified: 2021-09-20 description: BackgroundThere are several ecological studies, but few studies of the prevalence of SARS-COV-2 at high altitude. We aimed to estimate the population-based seroprevalence of SARS-COV-2 in three settings of Cusco at the end of the first wave among adults.MethodsA population-based survey was conducted in September 2020, in three settings in the region of Cusco: (1) Cusco city at 3,300 meters above the sea level (m.a.s.l.), (2) the periphery of Cusco (Santiago, San Jerónimo, San Sebastián, and Wanchaq) at 3,300 m.a.s.l., and (3) Quillabamba city, located at 1,050 m.a.s.l. People aged ≥ 18 years within a family unit were included. The diagnosis of SARS-CoV-2 infection was based on identifying anti- SARS-CoV-2 total antibodies (IgM and IgG) in serum using the Elecsys Anti-SARS-CoV-2 chemiluminescence test.ResultsWe enrolled 1924 participants from 712 families. Of the total, 637 participants were anti-SARS-CoV-2 seropositive. Seroprevalence was 38.8% (95% CI [33.4%–44.9%]) in Cusco city, 34.9% (95% CI [30.4%–40.1%]) in the periphery of Cusco, and 20.3% (95% CI [16.2%–25.6%]) in Quillabamba. In 141 families (19.8%; 95% CI [17.0%–22.8%]) the whole members were positive to the test. Living with more than three persons in the same house, a positive COVID-19 case at home, and a member who died in the last five months were factors associated with SARS-COV-2 seropositivity. Dysgeusia/dysosmia was the symptom most associated with seropositivity (aPR = 2.74, 95% CI [2.41–3.12]); whereas always wearing a face shield (aPR = 0. 73; 95% CI [0.60–0.89]) or a facial mask (aPR = 0.76, 95% CI [0.63–0. 92) reduced that probability.ConclusionsA great proportion of Cusco’s city inhabitants presented anti-SARS-CoV-2 antibodies at the end of the first wave, with significant differences between settings. Wearing masks and face shields were associated with lower rate of seropositivity; however, efforts must be made to sustain them over time since there is still a high proportion of susceptible people. creator: Charles Huamaní creator: Lucio Velásquez creator: Sonia Montes creator: Ana Mayanga-Herrera creator: Antonio Bernabé-Ortiz uri: https://doi.org/10.7717/peerj.12149 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Huamani et al. title: Caution ahead: reassessing the functional morphology of the respiratory organs in amphibious snails link: https://peerj.com/articles/12161 last-modified: 2021-09-20 description: After a long hiatus, interest in the morphology of the respiratory organs in apple snails (Ampullariidae, Caenogastropoda) and its functional and evolutionary bearings is making a comeback. The variability in the morphology of the gill and lung and its link to different lifestyles and patterns of air dependency within the Ampullariidae make research on the morphology of the respiratory organs particularly suitable for approaching the evolution of terrestriality in animals. Additionally, morphology is a valuable source of hypotheses regarding the several functions the ampullariid respiratory organs have besides respiration. However, this is an underexplored field that only recently has incorporated ultrastructural and three-dimension visualization tools and in which more research is much needed, particularly, comparisons between species representing the diversity within the Ampullariidae. In this paper, I examine Mueck, Deaton & Lee’s (2020a) assessment of the morphology of the gill and lung of Pomacea maculata and compare it with earlier and contemporary studies on other ampullariid species. I show that Mueck, Deaton & Lee’s paper combines significant morphological misinterpretations, conceptual and terminological mistakes, and crucial literature omissions. I also reinterpret their results and point to the similarities and differences between them and available data on other ampullariids. creator: Guido I. Prieto uri: https://doi.org/10.7717/peerj.12161 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Prieto title: Isokinetic force-power profile of the shoulder joint in males participating in CrossFit training and competing at different levels link: https://peerj.com/articles/11643 last-modified: 2021-09-17 description: BackgroundAs participants who engage in CrossFit training and competition perform a large volume of high intensity overhead activities, injuries to the shoulder are one of the most common in this sport. Previous research in other sports has indicated that the isokinetic force power profile of the shoulder joint (IPSJ) rotator muscles may assist in the prediction of shoulder injury.AimTherefore, the objective of this study was to determine the IPSJ in males engaged in CrossFit training at different competitive levels.MethodsIn a cross-sectional study design, participants (age, 24.1 ± 2.7 years) classified as ‘beginner’ (n = 6), ‘intermediate’ (n = 7) or ‘advanced’ (n = 9) provided informed consent to participate in this study. The IPSJ assessment involved rotational and diagonal movements, including internal and external shoulder rotator muscles, at both 180°.s−1 and 300°.s−1. The variables analysed were peak torque/body mass (%), mean power (W) and the external/internal peak torque/body mass ratio (%). A Kruskal–Wallis test was used to compare the IPSJ of the three groups, with Dunn’s test used for post-hoc analysis. The alpha level was set at p < 0.05.ResultsThe IPSJ showed greater torque and power values in those who competed at the advanced level as compared to those at a lower competitive level (i.e. intermediate, beginner). This was observed mainly for the internal rotation and internal diagonal movements at both 180°.s−1 and 300°.s−1. However, such differences between competitive levels were, in general, absent for the external rotation and external diagonal movements. Moreover, the participants from the advanced competitive level exhibited an imbalance of peak torque between the muscles responsible for the external–internal rotational and external-internal diagonal movements of the shoulder (i.e. peak torque external/internal ratio <66%), particularly in the dominant shoulder.ConclusionThese findings suggest greater development of the shoulder internal rotators and a higher probability of shoulder injury in CrossFit athletes at the advanced competitive level. Based on these results, participants engaged in CrossFit training and competition may wish to increase the volume of training for the shoulder external rotator muscles to complement the large increases in shoulder internal rotator strength that occur as a part of their regular training regimes. creator: Maximiliano A. Torres-Banduc creator: Daniel Jerez-Mayorga creator: Jason Moran creator: Justin W.L. Keogh creator: Rodrigo Ramírez-Campillo uri: https://doi.org/10.7717/peerj.11643 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Torres-Banduc et al. title: Soap, water, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): an ancient handwashing strategy for preventing dissemination of a novel virus link: https://peerj.com/articles/12041 last-modified: 2021-09-17 description: Public Health Agencies worldwide (World Health Organization, United States Centers for Disease Prevention & Control, Chinese Center for Disease Control and Prevention, European Centre for Disease Prevention and Control, etc.) are recommending hand washing with soap and water for preventing the dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. In this review, we have discussed the mechanisms of decontamination by soap and water (involving both removal and inactivation), described the contribution of the various components of formulated soaps to performance as cleansers and to pathogen inactivation, explained why adherence to recommended contact times is critical, evaluated the possible contribution of water temperature to inactivation, discussed the advantages of antimicrobial soaps vs. basic soaps, discussed the differences between use of soap and water vs. alcohol-based hand sanitizers for hand decontamination, and evaluated the limitations and advantages of different methods of drying hands following washing. While the paper emphasizes data applicable to SARS-CoV-2, the topics discussed are germane to most emerging and re-emerging enveloped and non-enveloped viruses and many other pathogen types. creator: M. Khalid Ijaz creator: Raymond W. Nims creator: Sarah de Szalay creator: Joseph R. Rubino uri: https://doi.org/10.7717/peerj.12041 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Ijaz et al. title: Effectiveness, tolerability and safety of Direct Acting Antivirals in Mexican individuals with Hepatitis C virus genotype-1 and previous pegylated interferon and ribavirin therapy link: https://peerj.com/articles/12051 last-modified: 2021-09-17 description: BackgroundDirect Acting Antivirals (DAAs) represent a large improvement in the treatment of chronic hepatitis C, resulting in <90% sustained virological response (SVR). There are no reports on the real-world DAA response for Mexico and few reports exist for Latin America. The aim of the study was to report SVR, and immediate benefits with the DAA treatments sofosbuvir, ledispavir, with/without ribavirin (SOF/LDV ± RBV) and ombitasvir, paritaprevir, ritonavir, dasabuvir with/without RBV (OBV/PTV/r/DSV ± RBV) in patients with viral genotype 1a or 1b, and who did not respond to previous peginterferon/ribavirin (PegIFNα2a+RBV) therapy.MethodsA descriptive, ambispective, longitudinal study was conducted. A cohort of 261 adult patients received PegIFNα2a+RBV therapy before 2014; 167 (64%) did not respond, 83 of these were subsequently treated with SOF/LDV ± RBV or OBV/PTV/r/DSV ± RBV. Child-Pugh-Score (CPS), Fibrosis-4 (FIB-4), and AST to Platelet Ratio Index (APRI) were evaluated before and after treatment.ResultsSVR with PegIFNα2a+RBV was 36%, and 97.5% with DAAs. CPS, FIB-4 and APRI improved significantly after DAA treatment, mainly because of liver transaminase reduction.ConclusionsDAA treatment showed excellent SVR rates in Mexican patients who had not responded to PegIFNα2a+RBV therapy. Improvement in CPS, FIB-4 and APRI without improvement in fibrosis was observed in cirrhotic and non-cirrhotic patients, as well as considerable reduction in liver transaminases, which suggests a reduction in hepatic necroinflammation. creator: Daniel Melendez-Mena creator: Miguel Angel Mendoza-Torres creator: Virginia Sedeño-Monge creator: Víctor Hugo García y García creator: Elain Rivera-García creator: Laura Sánchez-Reza creator: María del Carmen Baxin Domínguez creator: Belinda Guzmán-Flores creator: Ygnacio Martinez-Laguna creator: José Manuel Coronel Espinoza creator: Iván Galindo-Santiago creator: Juan Carlos Flores-Alonso creator: Verónica Vallejo-Ruiz creator: Paulina Cortes-Hernandez creator: Julio Reyes-Leyva creator: Francisca Sosa-Jurado creator: Gerardo Santos-López uri: https://doi.org/10.7717/peerj.12051 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Melendez-Mena et al. title: Assessment of Aleutian mink disease virus (AMDV) prevalence in feral American mink in Iceland. Case study of a pending epizootiological concern in Europe link: https://peerj.com/articles/12060 last-modified: 2021-09-17 description: BackgroundRecurring escapes or deliberate releases and subsequent infiltration or establishment of feral populations by individuals from fur farms have been commonly noted since the beginning of fur industry expansion. Once animals have invaded ecosystems adjacent to source farms escapees can change the demography of the feral populations through hybridization, outbreeding depression, competition and spreading of various pathogens which can decimate wild populations. In our study, we aimed to assess spread of Aleutian mink disease virus (AMDV) in the feral population of American mink (Neovison vison) in Iceland. The additional objective was to elucidate whether basic morpho-anatomical parameters (i.e., Fulton’s condition factor or spleen to body weight ratio) might be used as a preliminary indicator of AMDV infection.MethodsAmerican mink (n = 164) were captured by professional hunters in 8 regions of Iceland. The detection of AMDV in the spleen of male and female individuals was based on PCR amplification of an NS1 gene fragment.ResultsWe confirmed AMDV presence in 23.8% (n = 39) of collected samples with no significant difference in infection rate between males and females. Additionally, we revealed that the prevalence of virus in the feral population was higher closer to fur farms. However, the countrywide prevalence and direction of AMDV distribution needs to be further investigated. Comparison of condition indices in non-infected and infected animals showed significant deterioration of body and spleen parameters in the latter group. Therefore, the application of basic measurements of the American mink may be used to evaluate the health status of individuals in terms of pathogen infection.ConclusionsThe study shed a new light on prevalence and distribution of AMDV in the feral population of American mink in Iceland and the results might be successfully applied to develop models to infer dynamics of various pathogens, even those latently transmitted by disease-free animals. creator: Remigiusz Panicz creator: Piotr Eljasik creator: Jakub Skorupski creator: Przemysław Śmietana creator: Róbert A. Stefánsson creator: Menja von Schmalensee creator: Magdalena Szenejko uri: https://doi.org/10.7717/peerj.12060 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Panicz et al. title: A cross-sectional study of the nasal and fecal microbiota of sows from different health status within six commercial swine farms link: https://peerj.com/articles/12120 last-modified: 2021-09-17 description: BackgroundCull sows are a unique population on swine farms, often representing poor producing or compromised animals, and even though recent studies have reported that the microbiome is associated with susceptibility to diseases, the microbiome of the cull sow population has not been explored. The main objective of this study was to investigate whether there were differences in fecal and upper respiratory tract microbiota composition for groups of sows of different health status (healthy, cull, and compromised/ clinical sows) and from different farms (1 to 6).MethodsSix swine farms were visited once. Thirty individual fecal samples and nasal swabs were obtained at each farm and pooled by five across health status and farm. Samples underwent 16S rRNA gene amplicon sequencing and nasal and fecal microbiota were analyzed using QIIME2 v.2021.4.ResultsOverall, the diversity of the nasal microbiota was lower than the fecal microbiota (p < 0.01). No significant differences were found in fecal or nasal alpha diversity by sow’s health status or by farm. There were significant differences in nasal microbial composition by farm and health status (PERMANOVA, p < 0.05), and in fecal microbiota by farm (PERMANOVA, p < 0.05), but not by health status. Lastly, at the L7 level, there was one differentially abundant taxa across farms for each nasal and fecal pooled samples.DiscussionThis study provided baseline information for nasal and fecal microbiota of sows under field conditions, and results suggest that farm of origin can affect microbial diversity and composition. Furthermore, sow’s health status may have an impact on the nasal microbiota composition. creator: Andreia G. Arruda creator: Loic Deblais creator: Vanessa L. Hale creator: Christopher Madden creator: Monique Pairis-Garcia creator: Vishal Srivastava creator: Dipak Kathayat creator: Anand Kumar creator: Gireesh Rajashekara uri: https://doi.org/10.7717/peerj.12120 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Arruda et al. title: Exosomal hsa-miR-21-5p is a biomarker for breast cancer diagnosis link: https://peerj.com/articles/12147 last-modified: 2021-09-17 description: PurposeBreast cancer (BC) is characterized by concealed onset, delayed diagnosis, and high fatality rates making it particularly dangerous to patients’ health. The purpose of this study was to use comprehensive bioinformatics analysis and experimental verification to find a new biomarker for BC diagnosis.MethodsWe comprehensively analyzed microRNA (miRNA) and mRNA expression profiles from the Gene Expression Omnibus (GEO) and screened out differentially-expressed (DE) miRNAs and mRNAs. We used the miRNet website to predict potential DE-miRNA target genes. Using the Database for Annotation, Visualization and Integrated Discovery (DAVID), we performed Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on overlapping potential target genes and DE-mRNAs. The protein-protein interaction (PPI) network was then established. The miRNA-mRNA regulatory network was constructed using Cytoscape and the analysis results were visualized. We verified the expression of the most up-regulated DE-miRNA using reverse transcription and a quantitative polymerase chain reaction in BC tissue. The diagnostic value of the most up-regulated DE-miRNA was further explored across three levels: plasma-derived exosomes, cells, and cell exosomes.ResultsOur comprehensive bioinformatics analysis and experimental results showed that hsa-miR-21-5p was significantly up-regulated in BC tissue, cells, and exosomes. Our results also revealed that tumor-derived hsa-miR-21-5p could be packaged in exosomes and released into peripheral blood. Additionally, when evaluating the diagnostic value of plasma exosomal hsa-miR-21-5p, we found that it was significantly up-regulated in BC patients. Receiver operating characteristic (ROC) analysis also confirmed that hsa-miR-21-5p could effectively distinguish healthy people from BC patients. The sensitivity and specificity were 86.7% and 93.3%, respectively.ConclusionThis study’s results showed that plasma exosomal hsa-miR-21-5p could be used as a biomarker for BC diagnosis. creator: Min Liu creator: Fei Mo creator: Xiaohan Song creator: Yun He creator: Yan Yuan creator: Jiaoyan Yan creator: Ye Yang creator: Jian Huang creator: Shu Zhang uri: https://doi.org/10.7717/peerj.12147 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Liu et al. title: Variability in deer diet and plant vulnerability to browsing among forests with different establishment years of sika deer link: https://peerj.com/articles/12165 last-modified: 2021-09-17 description: Increased ungulate browsing alters the composition of plant communities and modifies forest ecosystems worldwide. Ungulates alter their diet following changes in availability of plant species; however, we know little about how browse selection and plant community composition change with different stages of deer establishment. Here, we provide insight into this area of study by combining multiple approaches: comparison of the understory plant community, analysis of records of browsing damage, and DNA barcoding of sika deer feces at 22 sites in forests in northern Japan varying in when deer were first established. The coverage of vegetation and number of plant species were only lower at sites where deer were present for more than 20 years, while the difference in plant coverage among deer establishment years varied among plant species. Deer diet differed across establishment years, but was more affected by the site, thereby indicating that food selection by deer could change over several years after deer establishment. Plant life form and plant architecture explained the difference in plant coverage across establishment years, but large variability was observed in deer diet within the two categories. Integrating these results, we categorized 98 plant taxa into six groups that differed in vulnerability to deer browsing (degree of damage and coverage). The different responses to browsing among plant species inferred from this study could be a first step in predicting the short- and long-term responses of forest plant communities to deer browsing. creator: Yuzu Sakata creator: Nami Shirahama creator: Ayaka Uechi creator: Kunihiro Okano uri: https://doi.org/10.7717/peerj.12165 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Sakata et al. title: Model for finding the number of honey bee colonies needed for the optimal foraging process in a specific geographical location link: https://peerj.com/articles/12178 last-modified: 2021-09-17 description: Finding a proper location for a bee apiary is a crucial task for beekeepers and especially for travelling beekeepers. Normally beekeepers choose an appropriate apiary location based on their previous experience and sometimes the location may not be optimal for the bee colonies. This can be explained by different flowering periods, variation of resources at the known fields, as well as other factors. In addition it is very challenging to evaluate how many bee colonies should be placed in one geographical location for an optimal nectar foraging process. This research presents a model for finding the number of honey bee colonies needed for the optimal foraging process in the specific location, taking into account several assumptions. Authors propose to take into account potential field productivity, possible chemical contamination, surroundings of the apiary. To run the model, several steps have to be completed, starting from the selection of area of interest, conversion to polygons for further calculations, defining the roads in the selected area. The outcome of the model number of colonies that should be placed is presented to the user. The Python language was used for the model development. The model can be extended to use additional factors and values to increase the precision of the evaluation. In addition, input from users (farmers, agricultural specialists, etc.) about external factors that can affect the number of bee colonies in the apiary can be taken into account. This work is conducted within the Horizon 2020 FET project HIVEOPOLIS (Nr.824069). creator: Olvija Komasilova creator: Vitalijs Komasilovs creator: Armands Kviesis creator: Aleksejs Zacepins uri: https://doi.org/10.7717/peerj.12178 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Komasilova et al. title: Evidence from the resurrected family Polyrhabdinidae Kamm, 1922 (Apicomplexa: Gregarinomorpha) supports the epimerite, an attachment organelle, as a major eugregarine innovation link: https://peerj.com/articles/11912 last-modified: 2021-09-16 description: BackgroundGregarines are a major group of apicomplexan parasites of invertebrates. The gregarine classification is largely incomplete because it relies primarily on light microscopy, while electron microscopy and molecular data in the group are fragmentary and often do not overlap. A key characteristic in gregarine taxonomy is the structure and function of their attachment organelles (AOs). AOs have been commonly classified as “mucrons” or “epimerites” based on their association with other cellular traits such as septation. An alternative proposal focused on the AOs structure, functional role, and developmental fate has recently restricted the terms “mucron” to archigregarines and “epimerite” to eugregarines.MethodsLight microscopy and scanning and transmission electron microscopy, molecular phylogenetic analyses of ribosomal RNA genes.ResultsWe obtained the first data on fine morphology of aseptate eugregarines Polyrhabdina pygospionis and Polyrhabdina cf. spionis, the type species. We demonstrate that their AOs differ from the mucron in archigregarines and represent an epimerite structurally resembling that in other eugregarines examined using electron microscopy. We then used the concatenated ribosomal operon DNA sequences (SSU, 5.8S, and LSU rDNA) of P. pygospionis to explore the phylogeny of eugregarines with a resolution superior to SSU rDNA alone. The obtained phylogenies show that the Polyrhabdina clade represents an independent, deep-branching family in the Ancoroidea clade within eugregarines. Combined, these results lend strong support to the hypothesis that the epimerite is a synapomorphic innovation of eugregarines. Based on these findings, we resurrect the family Polyrhabdinidae Kamm, 1922 and erect and diagnose the family Trollidiidae fam. n. within the superfamily Ancoroidea Simdyanov et al., 2017. Additionally, we re-describe the characteristics of P. pygospionis, emend the diagnoses of the genus Polyrhabdina, the family Polyrhabdinidae, and the superfamily Ancoroidea. creator: Gita G. Paskerova creator: Tatiana S. Miroliubova creator: Andrea Valigurová creator: Jan Janouškovec creator: Magdaléna Kováčiková creator: Andrei Diakin creator: Yuliya Ya. Sokolova creator: Kirill V. Mikhailov creator: Vladimir V. Aleoshin creator: Timur G. Simdyanov uri: https://doi.org/10.7717/peerj.11912 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Paskerova et al. title: GnRH or estradiol benzoate combination with CIDR improves in-vivo embryo production in bovines (Bos indicus and Bos taurus) under subtropics link: https://peerj.com/articles/12077 last-modified: 2021-09-16 description: Multiple Ovulation and Embryo Transfer (MOET) technology is a potential technique to upgrade livestock species’ genetics. The varied response to super-stimulatory treatments remains one of the limiting factors to this technology’s widespread use. The present study was aimed to improve the superovulation response and in-vivo embryo production by using controlled internal drug release (CIDR)-GnRH or CIDR-EB (Estradiol Benzoate) along with conventional superovulation protocol in Holstein Frisian (HF): Bos taurus; n = 42) and Crossbred (XB: Cholistani (Bos indicus) × HF; n = 28) cows. In the CIDR-GnRH/CIDR-EB treatment, CIDR was implanted in the cows after confirming the presence of a corpus luteum (CL) on the 8th day after estrus. 2 ml GnRH (Lecirelin acetate 0.0262 mg/ml) or 2 mg EB was also administered in CIDR-GnRH/CIDR-EB groups, respectively. Both groups were given super-stimulatory treatment from the 11th day after estrus (FSH in tapering doses twice a day for four consecutive days). On day 13, two doses of 2 ml prostaglandin (75 µg/ml of dextrorotatory cloprostenol) were administered (am: pm), and CIDR was removed the following day. Two artificial inseminations (AI) of the cows were performed (12 h apart) on the 15th day. No CIDR and GnRH/E.B were given in the control group, but the remaining superovulation protocol was the same. Later on, seven days after the first AI, non-surgical embryo flushing was done. The transferable embryos produced from three different superovulation protocols were then transferred into the recipient cows (n = 90) for determining their fertility. Statistical analysis revealed that the number of super-estrus follicles (SEF), multiple corpora lutea (MCL), ovulation/fertilization percentage, fertilized structures recovered (FSR), and transferable embryos (TEs) remained significantly higher (p < 0.05), and days taken for return to estrus (RTE) after embryo collection remained significantly lower (p < 0.05) in CIDR-GnRH (n = 18) and CIDR-EB (n = 15) groups as compared to the control (n = 37). The comparison between XB and HF cows revealed that the TEs production in CIDR-GnRH (XB = 5 vs HF = 13) and CIDR-EB (XB = 6 vs HF = 9) based superovulation protocols were 11.60  ±  4.08 vs 04.31  ±  0.98 and 09.33  ±  1.78 vs 05.22  ±  1.36, respectively. TEs production in XB cows (n = 5) of the CIDR-GnRH group was significantly higher (11.60  ±  4.08) than other groups. On the other hand, the days taken for RTE after embryo collection remained significantly lower (p < 0.05) in HF cows of treatment groups. However, the fertility of TEs was neither affected significantly (p > 0.05) by the superovulation protocol used nor by breed differences among donor cows. In conclusion, using CIDR-GnRH or CIDR-EB along with conventional superovulation protocol may enhance the efficiency of MOET programs in cattle. Furthermore, XB donor cows demonstrated a better performance than HF donor cows under subtropical conditions. creator: Khalid Mahmood creator: Muhammad Zahid Tahir creator: Mahboob Ahmad Butt creator: Shazia Mansoor Qureshi creator: Amjad Riaz uri: https://doi.org/10.7717/peerj.12077 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Mahmood et al. title: Determining relevant traits for selecting landrace accessions of Phaseolus lunatus L. for insect resistance link: https://peerj.com/articles/12088 last-modified: 2021-09-16 description: Plant-insect interactions are a determining factor for sustainable crop production. Although plants can resist or tolerate herbivorous insects to varying degrees, even with the use of pesticides, insects can reduce plant net productivity by as much as 20%, so sustainable strategies for pest control with less dependence on chemicals are needed. Selecting plants with optimal resistance and photosynthetic traits can help minimize damage and maintain productivity. Here, 27 landrace accessions of lima beans, Phaseolus lunatus L., from the Yucatan Peninsula were evaluated in the field for morphological resistance traits, photosynthetic characteristics, insect damage and seed yield. Variation was found in physical leaf traits (number, area, and dry mass of leaves; trichome density, specific leaf thickness and hardness) and in physiological traits (photosynthetic rate, stomatal conductance, intercellular carbon, water-use efficiency, and transpiration). Five accessions (JMC1325, JMC1288, JMC1339, JMC1208 and JMC1264) had the lowest index for cumulative damage with the highest seed yield, although RDA analysis uncovered two accessions (JMC1339, JMC1288) with strong positive association of seed yield and the cumulative damage index with leaf production, specific leaf area (SLA) and total leaf area. Leaf traits, including SLA and total leaf area are important drivers for optimizing seed yield. This study identified 12 important morphological and physiological leaf traits for selecting landrace accessions of P. lunatus for high yields (regardless of damage level) to achieve sustainable, environmentally safe crop production. creator: Roberto Rafael Ruiz-Santiago creator: Horacio Salómon Ballina-Gómez creator: Esau Ruiz-Sánchez creator: Jaime Martínez-Castillo creator: René Garruña-Hernández creator: Rubén Humberto Andueza-Noh uri: https://doi.org/10.7717/peerj.12088 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Ruiz-Santiago et al. title: miR-1293 acts as a tumor promotor in lung adenocarcinoma via targeting phosphoglucomutase 5 link: https://peerj.com/articles/12140 last-modified: 2021-09-16 description: BackgroundLung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer. Studies have found that miR-1293 is related to the survival of LUAD patients. Unfortunately, its role in LUAD remains not fully clarified.MethodsmiR-1293 expression and its association with LUAD patients’ clinical characteristics were analyzed in TCGA database. Also, miR-1293 expression was detected in LUAD cell lines. Cell viability, migration, invasion and expression of MMP2 and MMP9 were measured in LUAD cells following transfection with miR-1293 mimic or antagomir. Phosphoglucomutase (PGM) 5 was identified to be negatively related to miR-1293 in LUAD patients in TCGA database, and their association was predicated by Targetscan software. Hence, we further verified the relationship between miR-1293 and PGM5. Additionally, the effect and mechanism of miR-1293 were validated in a xenograft mouse model.ResultsWe found miR-1293 expression was elevated, but PGM5 was decreased, in LUAD patients and cell lines. Higher miR-1293 expression was positively related to LUAD patients’ pathologic stage and poor overall survival. miR-1293 mimic significantly promoted, whereas miR-1293 antagomir suppressed the viability, migration, invasion, and expression of MMP2 and MMP9 in LUAD cells. PGM5 was a target of miR-1293. Overexpression of PGM5 abrogated the effects of miR-1293 on the malignant phenotypes of LUAD cells. Administration of miR-1293 antagomir reduced tumor volume and staining of Ki-67 and MMP9, but elevated PGM5 expression in vivo.ConclusionsmiR-1293 promoted the proliferation, migration and invasion of LUAD cells via targeting PGM5, which indicated that miR-1293 might serve as a potential therapeutic target for LUAD patients. creator: Bing Chen creator: Shiya Zheng creator: Feng Jiang uri: https://doi.org/10.7717/peerj.12140 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Chen et al. title: DNMT family induces down-regulation of NDRG1 via DNA methylation and clinicopathological significance in gastric cancer link: https://peerj.com/articles/12146 last-modified: 2021-09-16 description: BackgroundAberrant DNA methylation of tumor suppressor genes is a common event in the development and progression of gastric cancer (GC). Our previous study showed NDRG1, which could suppress cell invasion and migration, was frequently down-regulated by DNA methylation of its promoter in GC.Purpose and MethodsTo analyze the relationship between the expression and DNA methylation of NDRG1 and DNA methyltransferase (DNMT) family. We performed a comprehensive comparison analysis using 407 patients including sequencing analysis data of GC from TCGA.ResultsNDRG1 was down-regulated in GC, and was negatively correlative to DNMT1 (r = −0.11, p = 0.03), DNMT3A (r = −0.10, p = 0.01), DNMT3B (r = −0.01, p = 0.88), respectively, whereas the DNA methylation of NDRG1 was positively correlative to DNMT family (DNMT1 r = 0.20, p < 0.01; DNMT3A r = 0.26, p < 0.001; DNMT3B r = 0.03, p = 0.57, respectively). NDRG1 expression was significantly inverse correlated with invasion depth (p = 0.023), but DNMT1 was significantly positive correlated with invasion depth (p = 0.049). DNMT3B was significantly correlated with the degree of tumor cell differentiation (p = 0.030). However, there was no association between the expression of DNMT3A and clinicopathological features. The KM plotter showed that NDRG1 (HR = 0.95, 95% CI [0.8–1.12], p = 0.53) and DNMT1 (HR = 1.04, 95% CI [0.88–1.23], p = 0.67) had no association with prognosis of GC patients, while, DNMT3A (p = 0.0064) and DNMT3B (p = 0.00025) displayed significantly association. But the overall survival of high expression of NDRG1 tended to be prolonged.ConclusionThese data suggest that down-regulation of NDRG1expression in GC may be due to its promoter DNA methylation via DNMT family. The demethylating agent maybe a potential target drug for GC patients. creator: Xiaojing Chang creator: Jinguo Ma creator: Xiaoying Xue creator: Guohui Wang creator: Tianfang Yan creator: Linlin Su creator: Xuetao Han creator: Huandi Zhou creator: Liubing Hou uri: https://doi.org/10.7717/peerj.12146 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Chang et al. title: Antioxidants, inhibits the growth of foodborne pathogens and reduces nitric oxide activity in LPS-stimulated RAW 264.7 cells of nipa palm vinegar link: https://peerj.com/articles/12151 last-modified: 2021-09-16 description: Nipa palm vinegar (NPV) made from the sap of nipa palm (Nypa fruticans Wurmb.) has long been used as a local food seasoning and folk medicine. This study compared the bioactive compounds, antioxidant, in vitro anti-inflammatory and antimicrobial activities of three NPVs obtained from different plantations based on varied soil and water salinity levels, including fresh water NPV, brackish water NPV and saline water NPV. The analysis results revealed that total phenolic content of saline water NPV had statistically significantly lower than both fresh water and brackish water NPV (p < 0.0001). Furthermore percentage of acetic acid in brackish water NPV had statistically significantly lower than both fresh water and saline water. NPV (p = 0.002). Nevertheless, total flavonoid and pH, were not significantly different (p = 0.144 and 0.066, respectively). The antioxidant activities using three ABTS, DPPH and FRAP methods displayed similar patterns, in which saline water NPV showed the highest antioxidant activities, followed by brackish water and fresh water NPV, respectively. Antimicrobial activity was examined for seven enteropathogenic bacteria. The tested NPVs were found inhibitive against all test cultures with a minimum inhibitory concentration (MIC) of ≤ 7.8 µL/mL. The cytotoxicity of the NPV obtained from different plantations by MTT assay revealed low cytotoxicity. Anti-inflammatory activity was also carried out through the inhibition of nitric oxide production. The fresh water NPV exhibited the highest anti-inflammatory activity with IC50 17.59 ± 0.17 µL/mL, followed by saline and brackish water NPV with IC50 18.12 ± 0.49 and 28.29 ± 2.64 µL/mL, respectively. The findings indicated that NPV from different soil salinities could potentially be natural functional food and developed to antimicrobial and anti-inflammatory medicinal agents with safety. creator: Wilaiwan Senghoi creator: Wiyada Kwanhian Klangbud uri: https://doi.org/10.7717/peerj.12151 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Senghoi and Klangbud title: Spinal curvatures of yoga practitioners compared to control participants—a cross-sectional study link: https://peerj.com/articles/12185 last-modified: 2021-09-16 description: PurposeThe angles of thoracic kyphosis and lumbar lordosis determine the spinal alignment in the sagittal plane. The aim of this study was to compare the thoracic kyphosis and lumbar lordosis of male and female yoga practitioners with non-practicing participants and to determine the possible dependencies between sagittal spinal curvatures and somatic parameters, time spent on yoga exercise, and undertaking other physical activities in yoga practitioners.MethodsThe study involved 576 women and 91 men ages 18–68 years (mean = 38.5 ± 9) who were practicing yoga, and 402 women and 176 men ages 18–30 years (mean = 20.2 ± 1.3) as a control group. The angles of thoracic kyphosis and lumbar lordosis were measured using a Plurimeter-V gravity inclinometer.ResultsThe two-way ANOVA demonstrated the influence of group (p < .0001) and sex (p = .03) on the angle of thoracic kyphosis, as well as the influence of group (p < .0001) and sex (p < .0001) on the angle of lumbar lordosis. It was noted that yoga practitioners had less pronounced thoracic kyphosis and lumbar lordosis and were more often characterized by normal or smaller thoracic kyphosis and lumbar lordosis than students from the control group. In yoga practitioners, the angle of thoracic kyphosis was positively correlated with age, body mass, BMI, and undertaking other forms of physical activity. The angle of lumbar lordosis was negatively correlated with body height and body mass.ConclusionsThe results suggest that yoga exercises can affect the shape of the anterior-posterior curves of the spine and may be an efficient training method for shaping proper posture in adults. creator: Małgorzata Grabara uri: https://doi.org/10.7717/peerj.12185 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Grabara title: Association of cerebral microvascular perfusion and diffusion dynamics detected by intravoxel incoherent motion-diffusion weighted imaging with initial neurological function and clinical outcome in acute ischemic stroke link: https://peerj.com/articles/12196 last-modified: 2021-09-16 description: BackgroundThis work aimed to explore the association of cerebral microvascular perfusion and diffusion dynamics measured by intravoxel incoherent motion (IVIM) imaging with initial neurological function and clinical outcome in acute stroke.MethodsIn total, 39 patients were assessed with admission National Institutes of Health Stroke Scale (NIHSS) and day-90 modified Rankin Scale (mRS). The parametrical maps of IVIM were obtained, including apparent diffusion coefficient (ADC), pseudo-diffusion coefficient (D*), true diffusion coefficient (D) and perfusion fraction (f). The fD* was the product of f and D*. Moreover, the ratios of lesioned/contralateral parameters (rADC, rD, rD*, rf and rfD*) were also obtained. The differences of these parameters between the poor outcome group and good outcome group were evaluated. Partial correlation analysis was used to evaluate the correlations between the admission NIHSS/day-90 mRS and each parameter ratio, with lesion volumes controlled.ResultsThe ADC, D, D*, f and fD* values of lesions were significantly reduced than those of the contralateral regions. The rADC and rD were significantly decreased in the poor outcome group than good outcome group (all p < 0.01). With lesion volume controlled, rADC showed a weak negative correlation (r = −0.340, p = 0.037) and a notable negative correlation (r = −0.688, p < 0.001) with admission NIHSS score and day-90 mRS score, respectively. In addition, rD showed a strong negative correlation (r = −0.731, p < 0.001) with day-90 mRS score.ConclusionSignificant negative correlations were revealed between IVIM derived diffusion dynamics parameters and initial neurological function as well as clinical outcome for patients with acute ischemic stroke. IVIM can be therefore suggested as an effective non-invasive method for evaluating the acute ischemic stroke. creator: Fei Chen creator: Zhenyu Dai creator: Lizheng Yao creator: Congsong Dong creator: Haicun Shi creator: Weiqiang Dou creator: Wei Xing uri: https://doi.org/10.7717/peerj.12196 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Chen et al. title: Struo2: efficient metagenome profiling database construction for ever-expanding microbial genome datasets link: https://peerj.com/articles/12198 last-modified: 2021-09-16 description: Mapping metagenome reads to reference databases is the standard approach for assessing microbial taxonomic and functional diversity from metagenomic data. However, public reference databases often lack recently generated genomic data such as metagenome-assembled genomes (MAGs), which can limit the sensitivity of read-mapping approaches. We previously developed the Struo pipeline in order to provide a straight-forward method for constructing custom databases; however, the pipeline does not scale well enough to cope with the ever-increasing number of publicly available microbial genomes. Moreover, the pipeline does not allow for efficient database updating as new data are generated. To address these issues, we developed Struo2, which is >3.5 fold faster than Struo at database generation and can also efficiently update existing databases. We also provide custom Kraken2, Bracken, and HUMAnN3 databases that can be easily updated with new genomes and/or individual gene sequences. Efficient database updating, coupled with our pre-generated databases, enables “assembly-enhanced” profiling, which increases database comprehensiveness via inclusion of native genomic content. Inclusion of newly generated genomic content can greatly increase database comprehensiveness, especially for understudied biomes, which will enable more accurate assessments of microbiome diversity. creator: Nicholas D. Youngblut creator: Ruth E. Ley uri: https://doi.org/10.7717/peerj.12198 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Youngblut and Ley title: Investigation and molecular identification of Eimeria sp. sampled from captive forest musk deer link: https://peerj.com/articles/11751 last-modified: 2021-09-15 description: Forest musk deer (Moschus berezovskii) is an endangered, protected species in China. Intestinal coccidiosis is a significant problem for captive forest musk deer. However, there are few reports on the prevalence and molecular characteristics of Eimeria sp. in forest musk deer. We sought to investigate the prevalence of Eimeria sp. in forest musk deer in the Sichuan and Shaanxi provinces in China. We also investigated the molecular characteristics of Eimeria sp. by analyzing the 18S rRNA gene. We collected a total of 328 fecal samples from forest musk deer on seven farms throughout the Sichuan and Shaanxi provinces. We extracted this parasite’s DNA and used this as a template for nested PCR amplification. The 18S rRNA gene fragment was associated with the plasmid vector, and these products were introduced into Escherichia coli (DH5α). The cultured bacterial solution was used as a PCR reaction template for identification purposes. We collected 328 fecal samples from forest musk deer in Lixian (n = 54), Maoxian (n = 52), Ma’erkang (n = 49), Dujiangyan (n = 55), Hanyuan (n = 41), Luding (n = 36) and Weinan (n = 41). One hundred ninety-eight (60.37%) fecal samples tested positive for Eimeria sp. . In our analysis of the 18S rRNA gene we found 34 types of Eimeria sp. with a similarity of 90.5–100%. We constructed a phylogenetic tree based on the parasite’s 18S rRNA gene sequence. Our findings indicated that the Eimeria sp. that parasitized the intestinal tract of forest musk deer was closely related to Eimeria alabamensis from Bos taurus and Eimeria ahsata from Ovis aries. To the best of our knowledge, ours was the first investigation and molecular identification of Eimeria sp. sampled from captive forest musk deer in China. Our results provide epidemiological data for the monitoring and prevention of Eimeria sp. in captive forest musk deer. creator: Ziwei Ren creator: Dong Yu creator: Wei Zhao creator: Yan Luo creator: Jianguo Cheng creator: Yin Wang creator: Zexiao Yang creator: Xueping Yao creator: Wei Yang creator: Xi Wu creator: Yimeng Li uri: https://doi.org/10.7717/peerj.11751 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Ren et al. title: Public perception of ecosystem services provided by the Mediterranean mussel Mytilus galloprovincialis related to anthropogenic activities link: https://peerj.com/articles/11975 last-modified: 2021-09-15 description: BackgroundMussels provide many ecosystem services as habitat, food, water filtration and recreational fishing. However, mussels are vulnerable to anthropogenic pressures such as harvesting or trampling, among others. In this frame, it would be paramount to engage society in marine conservation and improving its awareness about environmental policies. The first step lies in properly assessing what is the perception and concerns of society about marine ecosystems. Our study aims to fill this gap by examining public perception of services provided by Mytilus galloprovincialis, its state of conservation and the factors (including anthropogenic activities) shaping mussel beds.MethodsThis study is based on a face-to-face survey consisting of seven open-ended and seven multiple-choice questions of 404 people conducted in 2019 at different shores in the North Portuguese coast. The influence of respondent profile in terms of age, education, gender and place of residence was also assessed.ResultsMost of the participants in our survey (74%) considered that mussels contributed to human well-being and life quality; however, only 31% considered that mussels provide us with many benefits. Regarding the perceived state of mussel services, most of the respondents asserted that mussel services (purification of seawater, habitat, food for other species) worsened in the last 10 years. In contrast, the service as human food was perceived as in an identical state and scientific and traditional knowledge was the only service perceived in a better state. Concerning the state of mussel beds, most of the participants perceived it as good (45%) but a similar percentage (41%) asserted ignoring it. When considering the influence of different factors on mussel beds, only environmental management was considered as having a positive impact by a higher percentage of respondents. The majority of the participants considered that factors included in the questionnaire contributed to worsen mussel beds, ranging between 51% for coastal erosion and 90% for pollution. Education level and age were the main socio-economic factors driving public awareness about the importance of mussel services, its state of conservation and the factors shaping mussel beds.DiscussionResults showed that perception about the importance of mussels for human well-being and the quantity of delivered benefits increased with the education level. Moreover, older people perceived human food as the most important service offered by mussels. Therefore, our results suggest that mussels are mainly known as food resource; however, most of the people ignore their relevant ecological role and the many other benefits that mussels provide. Thus, it is necessary to actively engage society about importance of mussel beds. As M. galloprovincialis is a relevant economic resource, our data could improve the diffusion of knowledge among citizens, stakeholders and scientists, contributing to its sustainability. creator: Veiga Puri creator: Moreira Juan creator: Ramos-Oliveira Catarina creator: Sampaio Leandro creator: Marcos Rubal uri: https://doi.org/10.7717/peerj.11975 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 et al. title: The impact of peer review on the contribution potential of scientific papers link: https://peerj.com/articles/11999 last-modified: 2021-09-15 description: The peer-reviewing process has long been regarded as an indispensable tool in ensuring the quality of a scientific publication. While previous studies have tried to understand the process as a whole, not much effort has been devoted to investigating the determinants and impacts of the content of the peer review itself. This study leverages open data from nearly 5,000 PeerJ publications that were eventually accepted. Using sentiment analysis, Latent Dirichlet Allocation (LDA) topic modeling, mixed linear regression models, and logit regression models, we examine how the peer-reviewing process influences the acceptance timeline and contribution potential of manuscripts, and what modifications were typically made to manuscripts prior to publication. In an open review paradigm, our findings indicate that peer reviewers’ choice to reveal their names in lieu of remaining anonymous may be associated with more positive sentiment in their review, implying possible social pressure from name association. We also conduct a taxonomy of the manuscript modifications during a revision, studying the words added in response to peer reviewer feedback. This study provides insights into the content of peer reviews and the subsequent modifications authors make to their manuscripts. creator: Akira Matsui creator: Emily Chen creator: Yunwen Wang creator: Emilio Ferrara uri: https://doi.org/10.7717/peerj.11999 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Matsui et al. title: Post-fire dynamics of ectomycorrhizal fungal communities in a Scots pine (Pinus sylvestris L.) forest of Poland link: https://peerj.com/articles/12076 last-modified: 2021-09-15 description: BackgroundGlobal warming and drying have markedly enhanced in most forests the risk of fires across the world, which can affect the taxonomic and functional composition of key tree-associated organisms such as ectomycorrhizal (ECM) fungi. The present study was conducted to characterise the alterations in the extent of root ECM colonisation, the ECM fungal communities, and their exploration types (i.e., indicator of ECM soil foraging strategies) in regenerated pines within a burned site as compared with an unburned site (five years after the fire event) in the Forest District Myszyniec, Poland.MethodsTo assess the ECM fungal communities of burned and control sites, soil soil-root monoliths were collected from the study sites in September 2019. A total of 96 soil subsamples were collected for soil analysis and mycorrhizal assessment (6 trees × 2 sites × 4 study plots × 2 microsites (north and south) = 96 subsamples).ResultsThe percentage of root ECM colonisation was significantly lower in the burned site in comparison with the unburned (control) site. However, the ECM species richness did not differ between the control and burned sites. The identified ECM species in both sites were Imleria badia, Thelephora terrestris, Russula paludosa, R. badia, R. turci, R. vesca, Lactarius plumbeus, Phialocephala fortinii, and Hyaloscypha variabilis. The most frequent species in the burned and control sites were I. badia and T. terrestris, respectively. The relative abundances of contact, medium-distance smooth and long-distance exploration types in the burned site were significantly different from the control site, dominated by the medium-distance exploration type in both sites. The abundance of the long-distance exploration type in the burned site was markedly greater (27%) than that of the control site (14%), suggesting that the fire event had favoured this ECM foraging strategy. The results demonstrated that the fire led to reduced ECM colonisation of Scots pine trees in the burned site whereas the species richness was not affected, which can be attributed to degrees of fire-resistance in the ECM species, survival of ECM propagules in deeper soil layers, and/or continuous entry of spores/propagules of the ECM fungi from the adjacent forests via wind, water run-off or animals. creator: Jacek Olchowik creator: Dorota Hilszczańska creator: Marcin Studnicki creator: Tadeusz Malewski creator: Khalil Kariman creator: Zbigniew Borowski uri: https://doi.org/10.7717/peerj.12076 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Olchowik et al. title: Compositional distinction of gut microbiota between Han Chinese and Tibetan populations with liver cirrhosis link: https://peerj.com/articles/12142 last-modified: 2021-09-15 description: BackgroundLiver cirrhosis (LC) is caused by numerous chronic liver diseases and its complications are associated with qualitative and quantitative alterations of the gut microbiota. Previous studies have revealed the characteristics of gut microbiota in Han Chinese patients with LC and different compositions of gut microbiota were reported between the Tibetan and Han Chinese populations. This study was designed to evaluate the unique features of the gut microbiota of Tibetans and compare the differences of gut microbiota between Tibetan and Han Chinese patients with LC.MethodsThirty-six patients with liver cirrhosis and nineteen healthy volunteers, from both Tibetan and Han Chinese populations, were enrolled and fecal samples were collected for 16S rRNA gene sequencing analyses.ResultsSignificant differences were found in the gut microbiota of healthy volunteers and between Tibetan and Han Chinese patients with LC. In the Han Chinese patients with cirrhosis (HLC) group the relative abundances of the phylum Bacteroidetes was significantly reduced (P < 0.001), whereas in the Tibetan patients with cirrhosis (TLC) group Firmicutes and Actinobacteria were highly enriched (P = 0.01 and 0.03, respectively). At the genus level, the relative abundances of Anaerostipes (P < 0.001), Bifidobacterium (P = 0.03), and Blautia (P = 0.004) were prevalent, while Alloprevotella, Dorea, Prevotella_2, Prevotella_7 and Prevotella_9 were decreased in the TLC group compared to the HLC group (P < 0.01).ConclusionOur findings showed how the intestinal bacterial community shifted in Tibetan patients with cirrhosis. creator: Hui Huan creator: Tao Ren creator: Li Xu creator: Hong Hu creator: Chao Liu uri: https://doi.org/10.7717/peerj.12142 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Huan et al. title: Construction of competing endogenous RNA interaction network as prognostic markers in metastatic melanoma link: https://peerj.com/articles/12143 last-modified: 2021-09-15 description: Malignant melanoma (MM) is a malignant tumor originating from melanocytes, with high aggressiveness, high metastasis and extremely poor prognosis. MM accounts for 4% of skin cancers and 80% of mortality, and the median survival of patients with metastatic melanoma is only about 6 months, with a five-year survival rate of less than 10%. In recent years, the incidence of melanoma has gradually increased and has become one of the serious diseases that endanger human health. Competitive endogenous RNA (ceRNA) is the main model of the mechanism by which long chain non-coding RNAs (lncRNAs) play a regulatory role in the disease. LncRNAs can act as a “sponge”, competitively attracting small RNAs (micoRNAs; miRNAs), thus interfering with miRNA function, and affect the expression of target gene messenger RNAs (mRNAs), ultimately promoting tumorigenesis and progression. Bioinformatics analysis can identify potentially prognostic and therapeutically relevant differentially expressed genes in MM, finding lncRNAs, miRNAs and mRNAs that are interconnected through the ceRNA network, providing further insight into gene regulation and prognosis of metastatic melanoma. Weighted co-expression networks were used to identify lncRNA and mRNA modules associated with the metastatic phenotype, as well as the co-expression genes contained in the modules. A total of 17 lncRNAs, six miRNAs, and 11 mRNAs were used to construct a ceRNA interaction network that plays a regulatory role in metastatic melanoma patients. The prognostic risk model was used as a sorter to classify the survival prognosis of melanoma patients. Four groups of ceRNA interaction triplets were finally obtained, which miR-3662 might has potential implication for the treatment of metaststic melanoma patients, and futher experiments confirmed the regulating relationship and phenotype of this assumption. This study provides new targets to regulate metastatic process, predict metastatic potential and indicates that the miR-3662 can be used in the treatment of melanoma. creator: Zan He creator: Zijuan Xin creator: Yongfei Peng creator: Hua Zhao creator: Xiangdong Fang uri: https://doi.org/10.7717/peerj.12143 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 He et al. title: A randomized trial to evaluate the impact of copra meal hydrolysate on gastrointestinal symptoms and gut microbiome link: https://peerj.com/articles/12158 last-modified: 2021-09-15 description: The impact of copra meal hydrolysate (CMH) on gut health was assessed by conducting a double-blinded, placebo-controlled study. Sixty healthy adult participants, aged 18–40 years were assigned to daily consume 3 g of CMH, 5 g of CMH or placebo in the form of drink powder for 21 days. Consumption of CMH at 3 g/d improved defecating conditions by reducing stool size and also relieved flatulence and bloating symptoms. Fecal samples were collected serially at the baseline before treatment, after the treatment and after a 2-week washout period. The gut microbiomes were similar among the treatment groups, with microbial community changes observed within the groups. Intake of CMH at 3 g/d led to increase microbial diversity and richness. Reduction of the ratio between Firmicutes to Bacteroidetes was observed, although it was not significantly different between the groups. The 3 g/d CMH treatment increased beneficial microbes in the group of fiber-degrading bacteria, especially human colonic Bacteroidetes, while induction of Bifidobacteriaceae was observed after the washout period. Intake of CMH led to increase lactic acid production, while 3 g/d supplement promoted the present of immunoglobulin A (IgA) in stool samples. The 3 g daily dose of CMH led to the potentially beneficial effects on gut health for healthy individuals. creator: Witida Sathitkowitchai creator: Narissara Suratannon creator: Suttipun Keawsompong creator: Wanlapa Weerapakorn creator: Preecha Patumcharoenpol creator: Sunee Nitisinprasert creator: Massalin Nakphaichit uri: https://doi.org/10.7717/peerj.12158 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Sathitkowitchai et al. title: Novel ACE2 protein interactions relevant to COVID-19 predicted by evolutionary rate correlations link: https://peerj.com/articles/12159 last-modified: 2021-09-15 description: Angiotensin-converting enzyme 2 (ACE2) is the cell receptor that the coronavirus SARS-CoV-2 binds to and uses to enter and infect human cells. COVID-19, the pandemic disease caused by the coronavirus, involves diverse pathologies beyond those of a respiratory disease, including micro-thrombosis (micro-clotting), cytokine storms, and inflammatory responses affecting many organ systems. Longer-term chronic illness can persist for many months, often well after the pathogen is no longer detected. A better understanding of the proteins that ACE2 interacts with can reveal information relevant to these disease manifestations and possible avenues for treatment. We have undertaken an approach to predict candidate ACE2 interacting proteins which uses evolutionary inference to identify a set of mammalian proteins that “coevolve” with ACE2. The approach, called evolutionary rate correlation (ERC), detects proteins that show highly correlated evolutionary rates during mammalian evolution. Such proteins are candidates for biological interactions with the ACE2 receptor. The approach has uncovered a number of key ACE2 protein interactions of potential relevance to COVID-19 pathologies. Some proteins have previously been reported to be associated with severe COVID-19, but are not currently known to interact with ACE2, while additional predicted novel ACE2 interactors are of potential relevance to the disease. Using reciprocal rankings of protein ERCs, we have identified strongly interconnected ACE2 associated protein networks relevant to COVID-19 pathologies. ACE2 has clear connections to coagulation pathway proteins, such as Coagulation Factor V and fibrinogen components FGA, FGB, and FGG, the latter possibly mediated through ACE2 connections to Clusterin (which clears misfolded extracellular proteins) and GPR141 (whose functions are relatively unknown). ACE2 also connects to proteins involved in cytokine signaling and immune response (e.g. XCR1, IFNAR2 and TLR8), and to Androgen Receptor (AR). The ERC prescreening approach has elucidated possible functions for relatively uncharacterized proteins and possible new functions for well-characterized ones. Suggestions are made for the validation of ERC-predicted ACE2 protein interactions. We propose that ACE2 has novel protein interactions that are disrupted during SARS-CoV-2 infection, contributing to the spectrum of COVID-19 pathologies. creator: Austin A. Varela creator: Sammy Cheng creator: John H. Werren uri: https://doi.org/10.7717/peerj.12159 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Varela et al. title: Integrative bioinformatics and experimental analysis revealed down-regulated CDC42EP3 as a novel prognostic target for ovarian cancer and its roles in immune infiltration link: https://peerj.com/articles/12171 last-modified: 2021-09-15 description: Ovarian cancer is a significant clinical challenge as no effective treatments are available to enhance patient survival. Recently, N6-methyladenosine (m6A) RNA modification has been demonstrated to play a pivotal role in tumorigenesis and progression. However, the roles of m6A target genes in ovarian cancer haven’t been clearly illustrated. In this study, we presented a comprehensive bioinformatics and in vitro analysis to evaluate the roles of m6A target genes. Cell division cycle 42 effector protein 3 (CDC42EP3), one probable m6A target gene, was identified to be down-regulated in ovarian cancer tissues and cells. Meanwhile, quantitative PCR (qPCR) and western blot were used to confirm the down-regulated CDC42EP3 in ovarian cancer cells A2780 and TOV112D. The biological function of CDC42EP3 in ovarian cancer was further validated with several algorithms, such as PrognoScan, K-M plotter, LinkedOmics and TISIDB. These findings indicated that lower expression of CDC42EP3 was correlated with poor prognosis in patients with ovarian cancer. In addition, CDC42EP3 expression was significantly associated with a diverse range of tumor-infiltrating immune cells, including natural killer cells (NK), T central memory cells (Tcm), T gamma delta cells (Tgd), etc. Taken together, this study uncovered the potential roles of m6A target gene CDC42EP3 in the regulation of immune microenvironment in the ovarian cancer, and identified CDC42EP3 as a novel prognostic target. creator: Yuanliang Yan creator: Qiuju Liang creator: Zhijie Xu creator: Qiaoli Yi uri: https://doi.org/10.7717/peerj.12171 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Yan et al. title: The expansion and diversity of the CYP75 gene family in Vitaceae link: https://peerj.com/articles/12174 last-modified: 2021-09-15 description: The CYP75 gene family plays an important role in flavonoid biosynthesis in plants. Little is known about the evolution of the gene family within the grape family. Here, we extracted the CYP75 genes from transcriptome data of 15 grape species and 36 representative genomes from other plants to explore the evolutionary history of the CYP75 gene family in Vitaceae. The structure of the CYP75 protein sequences is highly conserved with the variation mainly occurring in the N terminal and the middle region. The evolutionary analyses suggested classifying the CYP75 gene family into three groups in Vitaceae, namely Vitaceae A1, Vitaceae A2 and Vitaceae B. The Vitaceae A1 and A2 belong to the CYP75A subfamily and the Vitaceae B belongs to the CYP75B subfamily. Within the Vitaceae A1, most Vitaceae taxa present only one copy of the CYP75A protein sequence except for Vitis vinifera with a high number of sequences, which might have originated through recent gene duplications after its split from the other species. Vitaceae A2 contain only CYP75A sequences from Vitaceae sister to one from Camellia sinensis, probably representing a relict lineage. The CYP75B proteins were found to be dominated in Vitaceae and other angiosperms. Our results provide important insights into understanding the evolutionary history of the CYP75 gene family in Vitaceae and other angiosperms. creator: Yang Xiao creator: Jun Wen creator: Ran Meng creator: Ying Meng creator: Qiang Zhou creator: Ze-Long Nie uri: https://doi.org/10.7717/peerj.12174 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Xiao et al. title: Identification and characterization of expression profiles of neuropeptides and their GPCRs in the swimming crab, Portunus trituberculatus link: https://peerj.com/articles/12179 last-modified: 2021-09-15 description: Neuropeptides and their G protein-coupled receptors (GPCRs) regulate multiple physiological processes. Currently, little is known about the identity of native neuropeptides and their receptors in Portunus trituberculatus. This study employed RNA-sequencing and reverse transcription-polymerase chain reaction (RT-PCR) techniques to identify neuropeptides and their receptors that might be involved in regulation of reproductive processes of P. trituberculatus. In the central nervous system transcriptome data, 47 neuropeptide transcripts were identified. In further analyses, the tissue expression profile of 32 putative neuropeptide-encoding transcripts was estimated. Results showed that the 32 transcripts were expressed in the central nervous system and 23 of them were expressed in the ovary. A total of 47 GPCR-encoding transcripts belonging to two classes were identified, including 39 encoding GPCR-A family and eight encoding GPCR-B family. In addition, we assessed the tissue expression profile of 33 GPCRs (27 GPCR-As and six GPCR-Bs) transcripts. These GPCRs were found to be widely expressed in different tissues. Similar to the expression profiles of neuropeptides, 20 of these putative GPCR-encoding transcripts were also detected in the ovary. This is the first study to establish the identify of neuropeptides and their GPCRs in P. trituberculatus, and provide information for further investigations into the effect of neuropeptides on the physiology and behavior of decapod crustaceans. creator: Shisheng Tu creator: Rui Xu creator: Mengen Wang creator: Xi Xie creator: Chenchang Bao creator: Dongfa Zhu uri: https://doi.org/10.7717/peerj.12179 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Tu et al. title: High-resolution prediction of American red squirrel in Interior Alaska: a role model for conservation using open access data, machine learning, GIS and LIDAR link: https://peerj.com/articles/11830 last-modified: 2021-09-14 description: American red squirrels (Tamiasciurus hudsonicus) are small mammals that are abundantly distributed throughout North America. Urbanization in the Anthropocene is now a global process, and squirrels live in affected landscapes. This leads to squirrels adjusting to human developments. Not much is known about the distribution of squirrels and squirrel middens near humans, especially not in the subarctic and sub-urbanized regions. Although this species is hunted, there are no real publicly available distribution and abundance estimates nor management plans and bag limits for squirrels in Alaska or in the United States known by us, except the endangered Mt. Graham squirrel. In general, insufficient squirrel conservation research is carried out; they are underrepresented in research and its literature. To further the science-based management for such species, this study aims to generate the first digital open access workflow as a generic research template for small mammal work including the latest machine learning of open source and high-resolution LIDAR data in an Open Source Geographic Information System (QGIS) and ArcGIS. Machine learning has proven to be less modeler biased and improve accuracy of the analysis outcome, therefore it is the preferred approach. This template is designed to be rapid, simple, robust, generic and effective for being used by a global audience. As a unique showcase, here a squirrel midden survey was carried out for two years (2016 and 2017). These squirrel middens were detected in a research area of 45,5 hectares (0,455 km2) in downtown Fairbanks, interior boreal forest of Alaska, U.S. Transect distances were geo-referenced with a GPS and adjusted to the visual conditions to count all squirrel middens within the survey area. Different layers of proximity to humans and habitat characteristics were assembled using aerial imagery and LIDAR data (3D data needed for an arboreal species like the red squirrels) consisting of a 3 × 3 m resolution. The layer data was used to train a predictive distribution model for red squirrel middens with machine learning. The model showed the relative index of occurrence (RIO) in a map and identified canopy height, distance to trails, canopy density and the distance to a lake, together, as the strongest predictors for squirrel midden distribution whereas open landscape and disturbed areas are avoided. It is concluded that squirrels select for high and dense forests for middens while avoiding human disturbance. This study is able to present a machine learning template to easily and rapidly produce an accurate abundance prediction which can be used for management implications. creator: Richard B. Robold creator: Falk Huettmann uri: https://doi.org/10.7717/peerj.11830 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Robold and Huettmann title: Glucosylceramide synthase regulates hepatocyte repair after concanavalin A-induced immune-mediated liver injury link: https://peerj.com/articles/12138 last-modified: 2021-09-14 description: BackgroundSphingolipids produce pleiotropic signaling pathways, and participate in the pathological mechanism of hepatocyte apoptosis and necrosis during liver injury. However, the role of glucosylceramide synthase (GCS)–key enzyme that catalyzes the first glycosylation step, in liver injury is still vague.MethodsAll experiments were conducted using 7–9-week-old pathogen-free male C57BL/6 mice. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected in murine models of liver disease, in addition to histological characterization of liver injuries. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression of the GCS, matrix metallopeptidase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1) genes. The GCS was observed through a fluorescence microscope, and the flow cytometry was used to detect hepatocyte apoptosis. The concentrations of serum IL-4, IL-6, and IL-10 were measured using enzyme-linked immune-sorbent assay (ELISA) kit. MMP-1 and TIMP-1 protein expression was measured via western blot (WB) analysis.ResultsCon A is often used as a mitogen to activate T lymphocytes and promote mitosis. A single dose of Con A injected intravenously will cause a rapid increase of ALT and AST, which is accompanied by the release of cytokines that cause injury and necrosis of hepatocytes. In this study, we successfully induced acute immune hepatitis in mice by Con A. Con A administration resulted in GCS upregulation in liver tissues. Moreover, the mice in the Con A group had significantly higher levels of ALT, AST, IL-4, IL-6, IL-10 and increased hepatocyte apoptosis than the control group. In contrast, all of the aforementioned genes were significantly downregulated after the administration of a GCS siRNA or Genz-123346 (i.e., a glucosylceramide synthase inhibitor) to inhibit the GCS gene. Additionally, the histopathological changes observed herein were consistent with our ALT, AST, IL-4, IL-6, and IL-10 expression results. However, unlike this, hepatocyte apoptosis has been further increased on the basis of the Con A group. Moreover, our qRT-PCR and WB results indicated that the expression of MMP-1 in the Con A group was significantly lower than that in the control group, whereas TIMP-1 exhibited the opposite trend. Conversely, MMP-1 expression in the GCS siRNA and Genz-123346 groups was higher than that in the Con A group, whereas TIMP-1 expression was lower.ConclusionsGCS inhibition reduces Con A-induced immune-mediated liver injury in mice, which may be due to the involvement of GCS in the hepatocyte repair process after injury. creator: Jian Gan creator: Qin Gao creator: Li Li Wang creator: Ai Ping Tian creator: Long Dong Zhu creator: Li Ting Zhang creator: Wei Zhou creator: Xiao Rong Mao creator: Jun Feng Li uri: https://doi.org/10.7717/peerj.12138 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Gan et al. title: Phylogenetic relationships of sucrose transporters (SUTs) in plants and genome-wide characterization of SUT genes in Orchidaceae reveal roles in floral organ development link: https://peerj.com/articles/11961 last-modified: 2021-09-13 description: Sucrose is the primary form of photosynthetically produced carbohydrates transported long distance in many plant species and substantially affects plant growth, development and physiology. Sucrose transporters (SUTs or SUCs) are a group of membrane proteins that play vital roles in mediating sucrose allocation within cells and at the whole-plant level. In this study, we investigated the relationships among SUTs in 24 representative plant species and performed an analysis of SUT genes in three sequenced Orchidaceae species: Dendrobium officinale, Phalaenopsis equestris, and Apostasia shenzhenica. All the SUTs from the 24 plant species were classified into three groups and five subgroups, subgroups A, B1, B2.1, B2.2, and C, based on their evolutionary relationships. A total of 22 SUT genes were identified among Orchidaceae species, among which D. officinale had 8 genes (DoSUT01-08), P. equestris had eight genes (PeqSUT01-08) and A. shenzhenica had 6 genes (AsSUT01-06). For the 22 OrchidaceaeSUTs, subgroups A, B2.2 and C contained three genes, whereas the SUT genes were found to have significantly expanded in the monocot-specific subgroup B2.1, which contained 12 genes. To understand sucrose partitioning and the functions of sucrose transporters in Orchidaceae species, we analyzed the water-soluble sugar content and performed RNA sequencing of different tissues of D. officinale, including leaves, stems, flowers and roots. The results showed that although the total content of water-soluble polysaccharides was highest in the stems of D. officinale, the sucrose content was highest in the flowers. Moreover, gene expression analysis showed that most of the DoSUTs were expressed in the flowers, among which DoSUT01,DoSUT07 and DoSUT06 had significantly increased expression levels. These results indicated that stems are used as the main storage sinks for photosynthetically produced sugar in D. officinale and that DoSUTs mainly function in the cellular machinery and development of floral organs. Our findings provide valuable information on sucrose partitioning and the evolution and functions of SUT genes in Orchidaceae and other species. creator: Yunzhu Wang creator: Yue Chen creator: Qingzhen Wei creator: Hongjian Wan creator: Chongbo Sun uri: https://doi.org/10.7717/peerj.11961 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Wang et al. title: Active behaviour of terrestrial caterpillars on the water surface link: https://peerj.com/articles/11971 last-modified: 2021-09-13 description: Most butterfly and moth larvae (Lepidoptera) are terrestrial. When terrestrial caterpillars accidentally fall into water, they may drown or be preyed upon by aquatic predators before they can safely reach land. However, how terrestrial caterpillars escape aquatic environments and predators remains unclear. In July 2018, we observed a terrestrial caterpillar actively moving forward on the surface of a pond in Japan until it successfully reached the shore. To further investigate this behaviour in terrestrial caterpillars, we experimentally placed larvae of 13 moth species (four families) on a water surface under laboratory and field conditions. All caterpillars floated. Larvae of seven species moved forward on the water surface, whereas those of six species did not. A total of two types of behaviour were observed; in Dinumma deponens, Hypopyra vespertilio, Spirama retorta, Laelia coenosa, Lymantria dispar (all Erebidae), and Naranga aenescens (Noctuidae), larvae swung their bodies rapidly from side to side to propel themselves along the water surface (i.e., undulatory behaviour); in contrast, larvae of Acosmetia biguttula (Noctuidae) rapidly moved the abdomen (posterior segments) up and down for propulsion along the water surface (i.e., flick behaviour). Although thoracic legs were not used for undulatory and flick behaviour, rapid movements of the abdomen were used to propel caterpillars on the water surface. We also observed that undulatory and flick behaviour on the water surface aided caterpillars in escaping aquatic predators under field conditions. In addition, we investigated the relationship between body size and undulatory behaviour on the water surface in the erebid S. retorta under laboratory conditions. The frequency and speed of forward movement on the water surface increased with body length. Together, these results show that the rapid movement of elongated bodies results in forward propulsion on the water surface, allowing some terrestrial caterpillars to avoid drowning or aquatic predators. We further suggested potential factors related to morphology, host plant habitat, and defensive behaviour that may have led to the acquisition of aquatic behaviour in terrestrial caterpillars. creator: Masakazu Hayashi creator: Shinji Sugiura uri: https://doi.org/10.7717/peerj.11971 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Hayashi and Sugiura title: Genome-scale identification of plant defensin (PDF) family genes and molecular characterization of their responses to diverse nutrient stresses in allotetraploid rapeseed link: https://peerj.com/articles/12007 last-modified: 2021-09-13 description: Plant defensins (PDFs), short peptides with strong antibacterial activity, play important roles in plant growth, development, and stress resistance. However, there are few systematic analyses on PDFs in Brassica napus. Here, bioinformatics methods were used to identify genome-wide PDFs in Brassica napus, and systematically analyze physicochemical properties, expansion pattern, phylogeny, and expression profiling of BnaPDFs under diverse nutrient stresses. A total of 37 full-length PDF homologs, divided into two subgroups (PDF1s and PDF2s), were identified in the rapeseed genome. A total of two distinct clades were identified in the BnaPDF phylogeny. Clade specific conserved motifs were identified within each clade respectively. Most BnaPDFs were proved to undergo powerful purified selection. The PDF members had enriched cis-elements related to growth and development, hormone response, environmental stress response in their promoter regions. GO annotations indicate that the functional pathways of BnaPDFs are mainly involved in cells killing and plant defense responses. In addition, bna-miRNA164 and bna-miRNA172 respectively regulate the expression of their targets BnaA2.PDF2.5 and BnaC7.PDF2.6. The expression patterns of BnaPDFs were analyzed in different tissues. BnaPDF1.2bs was mainly expressed in the roots, whereas BnaPDF2.2s and BnaPDF2.3s were both expressed in stamen, pericarp, silique, and stem. However, the other BnaPDF members showed low expression levels in various tissues. Differential expression of BnaPDFs under nitrate limitation, ammonium excess, phosphorus starvation, potassium deficiency, cadmium toxicity, and salt stress indicated that they might participate in different nutrient stress resistance. The genome-wide identification and characterization of BnaPDFs will enrich understanding of their molecular characteristics and provide elite gene resources for genetic improvement of rapeseed resistance to nutrient stresses. creator: Ying Liu creator: Ying-peng Hua creator: Huan Chen creator: Ting Zhou creator: Cai-peng Yue creator: Jin-yong Huang uri: https://doi.org/10.7717/peerj.12007 license: https://creativecommons.org/licenses/by-nc/4.0 rights: © 2021 Liu et al. title: Substrate type and palaeodepth do not affect the Middle Jurassic taxonomic diversity of crinoids link: https://peerj.com/articles/12017 last-modified: 2021-09-13 description: Crinoids are largely considered as good indicators for determining environmental conditions. They are robust proxies for inferring changes in salinity and sedimentation rate and for inferring substrate type. Some crinoid groups (e.g., certain comatulids, cyrtocrinids, millericrinids) have a depth preference, thus, making them useful for palaeodepth estimation. The hypotheses that crinoid distribution is substrate-dependent (rock type) or palaeodepth-dependent is tested here based on (a) archival Bathonian-Callovian (Middle Jurassic) crinoid occurrences from Poland and (b) newer finds from five boreholes from eastern Poland. Qualitative data suggests that isocrinids and cyclocrinids occur in both carbonate and siliciclastic rocks. The cyrtocrinids and roveacrinids occur within carbonate rocks, whereas the comatulids are exclusive to siliciclastics. In terms of palaeodepth, most crinoid groups dominate in shallow environments with the sole exception of cyrtocrinids, that are ubiquitous and occur in both shallow (near shore and shallow marine) and slightly deeper (deeper sublittoral to open shelf) settings. The occurrences of the cosmopolitan taxa, Chariocrinus andreae and Balanocrinus subteres (isocrinids), is independent of both substrate type and palaeodepth. Quantitative analyses (Analysis Of Variance; ANOVA) based on substrate type, i.e., substrate-dependency (claystones, sandstones and limestones), and palaeodepth i.e., palaeodepth-dependency (near shore, shallow-marine, mid-ramp and offshore), corroborate qualitative results. Statistical analysis suggest that the distribution of crinoids shows a strong substrate-dependency but not for palaeodepth, although very weak significance (low p value) is noted for near shore and shallow marine settings and crinoid distribution. creator: Mariusz A. Salamon creator: Anna Feldman-Olszewska creator: Sreepat Jain creator: Bruno B.M. Ferré creator: Karolina Paszcza creator: Bartosz J. Płachno uri: https://doi.org/10.7717/peerj.12017 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Salamon et al. title: Catostylus tagi (Class: Scyphozoa, Order: Discomedusae, Suborder: Rhizostomida, Family: Catostylidae) life cycle and first insight into its ecology link: https://peerj.com/articles/12056 last-modified: 2021-09-13 description: Jellyfish proliferations, which are conspicuous and natural events, cause blooms that may lead to severe consequences for anthropogenic activities and ecosystem structure and functioning. Although research during the last decade has focused on factors influencing the different jellyfish life stages, few species currently have their full life cycle known. In this context, we describe for the first time the developmental stages in the life cycle of Catostylus tagi, from planula to young medusa, reared in the laboratory. The species displays the typical Rhizostomida metagenetic life cycle. Mature scyphistomae display 16 tentacles and a total body length of 1.5 ± 0.2 mm. Only podocyst production and strobilation were observed. Strobilation, occurring continuously under laboratory conditions, was mainly polydisc. The eight-rayed typical ephyrae, with a total body diameter of 2.4 ± 0.4 mm at detachment, showed development typical of the Rhizostomida. As a first step in studying this species’ ecology, we also present preliminary assessments of: (i) the influence of different temperature and salinity regimes on planulae survival, settlement and metamorphosis and (ii) the effect of temperature and diet on asexual reproduction. The results showed a high tolerance of planulae to a wide range of salinities (15‰ to 25‰), while polyp development was significantly faster at higher temperature (20–25 °C). Strobilation onset was 2–3 times faster at 20 °C (10.6 ± 5.4 to 15 ± 6.6 day at various tested diet) than at 15 °C (32.2 ± 3 day). Feeding was a key factor as unfed polyps never underwent strobilation during the trial. Finally, we present the spatial and seasonal distribution of C. tagi in the Tagus estuary (Portugal) in 2019, showing its occurrence throughout the year (except in April), with most observations recorded on the northern shoreline. As C. tagi shows the ability to form blooms and a wide tolerance for temperature and salinity (for planulae and medusae stage), it is essential to understand its life cycle. creator: Sonia K.M. Gueroun creator: Tatiana M. Torres creator: Antonina Dos Santos creator: Nuno Vasco-Rodrigues creator: João Canning-Clode creator: Carlos Andrade uri: https://doi.org/10.7717/peerj.12056 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Gueroun et al. title: Identification and validation of hub genes for diabetic retinopathy link: https://peerj.com/articles/12126 last-modified: 2021-09-13 description: BackgroundDiabetic retinopathy (DR) is characterized by a gradually progressive alteration in the retinal microvasculature that leads to middle-aged adult acquired persistent blindness. Limited research has been conducted on DR pathogenesis at the gene level. Thus, we aimed to reveal novel key genes that might be associated with DR formation via a bioinformatics analysis.MethodsThe GSE53257 dataset from the Gene Expression Omnibus was downloaded for gene co-expression analysis. We identified significant gene modules via the Weighted Gene Co-expression Network Analysis, which was conducted by the Protein-Protein Interaction (PPI) Network via Cytoscape and from this we screened for key genes and gene sets for particular functional and pathway-specific enrichments. The hub gene expression was verified by real-time PCR in DR rats modeling and an external database.ResultsTwo significant gene modules were identified. Significant key genes were predominantly associated with mitochondrial function, fatty acid oxidation and oxidative stress. Among all key genes analyzed, six up-regulated genes (i.e., SLC25A33, NDUFS1, MRPS23, CYB5R1, MECR, and MRPL15) were highly and significantly relevant in the context of DR formation. The PCR results showed that SLC25A33 and NDUFS1 expression were increased in DR rats modeling group.ConclusionGene co-expression network analysis highlights the importance of mitochondria and oxidative stress in the pathophysiology of DR. DR co-expressing gene module was constructed and key genes were identified, and both SLC25A33 and NDUFS1 may serve as potential biomarker and therapeutic target for DR. creator: Li Peng creator: Wei Ma creator: Qing Xie creator: Baihua Chen uri: https://doi.org/10.7717/peerj.12126 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Peng et al. title: Sex- and stage-dependent expression patterns of odorant-binding and chemosensory protein genes in Spodoptera exempta link: https://peerj.com/articles/12132 last-modified: 2021-09-13 description: As potential molecular targets for developing novel pest management strategies, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) have been considered to initiate odor recognition in insects. Herein, we investigated the OBPs and CSPs in a major global crop pest (Spodoptera exempta). Using transcriptome analysis, we identified 40 OBPs and 33 CSPs in S. exempta, among which 35 OBPs and 29 CSPs had intact open reading frames. Sequence alignment indicated that 30 OBPs and 23 CSPs completely contained the conserved cysteines. OBPs of lepidopteran insects usually belonged to classical, minus-C, and plus-C groups. However, phylogenetic analyses indicated that we only identified 28 classical and seven minus-C OBPs in S. exempta, suggesting that we might have missed some typical OBPs in lepidopteran insects, probably due to their low expression levels. All of the CSPs from S. exempta clustered with the orthologs of other moths. The identification and expression of the OBPs and CSPs were well studied in insect adults by transcriptional analyses, and herein we used samples at different stages to determine the expression of OBPs and CSPs in S. exempta. Interestingly, our data indicated that several OBPs and CSPs were especially or more highly expressed in larvae or pupae than other stages, including three exclusively (SexeOBP13, SexeOBP16 and SexeCSP23) and six more highly (SexeOBP15, SexeOBP37, SexeCSP4, SexeCSP8, SexeCSP19, and SexeCSP33) expressed in larvae, two exclusively (SexeCSP6 and SexeCSP20) and three more highly (SexeOBP18, SexeCSP17, and SexeCSP26) expressed in pupae. Usually, OBPs and CSPs had both male- and female-biased expression patterns in adult antennae. However, our whole-body data indicated that all highly expressed OBPs and CSPs in adults were male-biased or did not differ, suggesting diverse OBP and CSP functions in insect adults. Besides identifying OBPs and CSPs as well as their expression patterns, these results provide a molecular basis to facilitate functional studies of OBPs and CSPs for exploring novel management strategies to control S. exempta. creator: Yonghao Dong creator: Tong Li creator: Jin Liu creator: Meixue Sun creator: Xingyu Chen creator: Yongjie Liu creator: Pengjun Xu uri: https://doi.org/10.7717/peerj.12132 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Dong et al. title: Biogeography rather than substrate type determines bacterial colonization dynamics of marine plastics link: https://peerj.com/articles/12135 last-modified: 2021-09-13 description: Since the middle of the 20th century, plastics have been incorporated into our everyday lives at an exponential rate. In recent years, the negative impacts of plastics, especially as environmental pollutants, have become evident. Marine plastic debris represents a relatively new and increasingly abundant substrate for colonization by microbial organisms, although the full functional potential of these organisms is yet to be uncovered. In the present study, we investigated plastic type and incubation location as drivers of marine bacterial community structure development on plastics, i.e., the Plastisphere, via 16S rRNA amplicon analysis. Four distinct plastic types: high-density polyethylene (HDPE), linear low-density polyethylene (LDPE), polyamide (PA), polymethyl methacrylate (PMMA), and glass-slide controls were incubated for five weeks in the coastal waters of four different biogeographic locations (Cape Verde, Chile, Japan, South Africa) during July and August of 2019. The primary driver of the coastal Plastisphere composition was identified as incubation location, i.e., biogeography, while substrate type did not have a significant effect on bacterial community composition. The bacterial communities were consistently dominated by the classes Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia, irrespective of sampling location or substrate type, however a core bacterial Plastisphere community was not observable at lower taxonomic levels. Overall, this study sheds light on the question of whether bacterial communities on plastic debris are shaped by the physicochemical properties of the substrate they grow on or by the marine environment in which the plastics are immersed. This study enhances the current understanding of biogeographic variability in the Plastisphere by including biofilms from plastics incubated in the previously uncharted Southern Hemisphere. creator: Ashley K. Coons creator: Kathrin Busch creator: Mark Lenz creator: Ute Hentschel creator: Erik Borchert uri: https://doi.org/10.7717/peerj.12135 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Coons et al. title: Habitat ephemerality affects the evolution of contrasting growth strategies and cannibalism in anuran larvae link: https://peerj.com/articles/12172 last-modified: 2021-09-13 description: Ephemeral streams are challenging environments for tadpoles; thus, adaptive features that increase the survival of these larvae should be favored by natural selection. In this study, we compared the adaptive growth strategies of Bombina orientalis (the oriental fire-bellied toad) tadpoles from ephemeral streams with those of such tadpoles from non-ephemeral streams. Using a common garden experiment, we tested the interactive effects of location (ephemeral vs. non-ephemeral), food availability, and growing density on larval period, weight at metamorphosis, and cannibalism. We found that tadpoles from ephemeral streams underwent a shorter larval period compared with those from non-ephemeral streams but that this difference was contingent on food availability. The observed faster growth is likely to be an adaptive response because tadpoles in ephemeral streams experience more biotic/abiotic stressors, such as desiccation risk and limited resources, compared with those in non-ephemeral streams, with their earlier metamorphosis potentially resulting in survival benefits. As a trade-off for their faster growth, tadpoles from ephemeral streams generally had a lower body weight at metamorphosis compared with those from non-ephemeral streams. We also found lower cannibalism rates among tadpoles from ephemeral streams, which can be attributed to the indirect fitness costs of cannibalizing their kin. Our study demonstrates how ephemeral habitats have affected the evolutionary change in cannibalistic behaviors in anurans and provides additional evidence that natural selection has mediated the evolution of growth strategies of tadpoles in ephemeral streams. creator: Dogeun Oh creator: Yongsu Kim creator: Sohee Yoo creator: Changku Kang uri: https://doi.org/10.7717/peerj.12172 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Oh et al. title: Capturing and analyzing pattern diversity: an example using the melanistic spotted patterns of leopard geckos link: https://peerj.com/articles/11829 last-modified: 2021-09-10 description: Animal color patterns are widely studied in ecology, evolution, and through mathematical modeling. Patterns may vary among distinct body parts such as the head, trunk or tail. As large amounts of photographic data is becoming more easily available, there is a growing need for general quantitative methods for capturing and analyzing the full complexity and details of pattern variation. Detailed information on variation in color pattern elements is necessary to understand how patterns are produced and established during development, and which evolutionary forces may constrain such a variation. Here, we develop an approach to capture and analyze variation in melanistic color pattern elements in leopard geckos. We use this data to study the variation among different body parts of leopard geckos and to draw inferences about their development. We compare patterns using 14 different indices such as the ratio of melanistic versus total area, the ellipticity of spots, and the size of spots and use these to define a composite distance between two patterns. Pattern presence/absence among the different body parts indicates a clear pathway of pattern establishment from the head to the back legs. Together with weak within-individual correlation between leg patterns and main body patterns, this suggests that pattern establishment in the head and tail may be independent from the rest of the body. We found that patterns vary greatest in size and density of the spots among body parts and individuals, but little in their average shapes. We also found a correlation between the melanistic patterns of the two front legs, as well as the two back legs, and also between the head, tail and trunk, especially for the density and size of the spots, but not their shape or inter-spot distance. Our data collection and analysis approach can be applied to other organisms to study variation in color patterns between body parts and to address questions on pattern formation and establishment in animals. creator: Tilmann Glimm creator: Maria Kiskowski creator: Nickolas Moreno creator: Ylenia Chiari uri: https://doi.org/10.7717/peerj.11829 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Glimm et al. title: Hepatitis C core antigen test as an alternative for diagnosing HCV infection: mathematical model and cost-effectiveness analysis link: https://peerj.com/articles/11895 last-modified: 2021-09-10 description: BackgroundThe cost and complexity of the polymerase chain reaction (PCR) test are barriers to diagnosis and treatment of hepatitis C virus (HCV) infection. We investigated the cost-effectiveness of testing strategies using antigen instead of PCR testing.MethodsWe developed a mathematical model for HCV to estimate the number of diagnoses and cases of liver disease. We compared the following testing strategies: antibody test followed by PCR in case of positive antibody (baseline strategy); antibody test followed by HCV-antigen test (antibody-antigen); antigen test alone; PCR test alone. We conducted cost-effectiveness analyses considering either the costs of HCV testing of infected and uninfected individuals alone (A1), HCV testing and liver-related complications (A2), or all costs including HCV treatment (A3). The model was parameterized for the country of Georgia. We conducted several sensitivity analyses.ResultsThe baseline scenario could detect 89% of infected individuals. Antibody-antigen detected 86% and antigen alone 88% of infected individuals. PCR testing alone detected 91% of the infected individuals: the remaining 9% either died or spontaneously recovered before testing. In analysis A1, the baseline strategy was not essentially more expensive than antibody-antigen. In analysis A2, strategies using PCR became cheaper than antigen-based strategies. In analysis A3, antibody-antigen was again the cheapest strategy, followed by the baseline strategy, and PCR testing alone.ConclusionsAntigen testing, either following a positive antibody test or alone, performed almost as well as the current practice of HCV testing. The cost-effectiveness of these strategies depends on the inclusion of treatment costs. creator: Maryam Sadeghimehr creator: Barbara Bertisch creator: Francesco Negro creator: Maia Butsashvili creator: Sonjelle Shilton creator: Irina Tskhomelidze creator: Maia Tsereteli creator: Olivia Keiser creator: Janne Estill uri: https://doi.org/10.7717/peerj.11895 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Sadeghimehr et al. title: Synopsis of Habenaria s.l. (Orchidaceae) in New Guinea and adjacent islands link: https://peerj.com/articles/12011 last-modified: 2021-09-10 description: A taxonomic synopsis of the orchid genus Habenaria in New Guinea and adjacent islands is presented. We confirmed the occurrence of 27 Habenaria species in study area. Sixteen of these are endemic and were not so far found outside New Guinea. Morphological characteristics and illustrations of floral segments of taxa are presented. One new species of Habenaria is described. Four neotypes are selected. An updated key to species groups and species occurring in the study area is provided. The importance of diagnostic floral characters in Habenaria is discussed. creator: Marta Kolanowska creator: Marta Kras creator: Sławomir Nowak creator: Dariusz L. Szlachetko uri: https://doi.org/10.7717/peerj.12011 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Kolanowska et al. title: A new species of Proceratophrys Miranda-Ribeiro, 1920 (Anura, Odontophrynidae) from Southern Amazonia, Brazil link: https://peerj.com/articles/12012 last-modified: 2021-09-10 description: Based on concordant differences in morphology, male advertisement call, and 16S mtDNA barcode distance, we describe a new species of Proceratophrys from southern Amazonia, in the states of Mato Grosso and Pará, Brazil. The new species is most similar to P. concavitympanum and P. ararype but differs from these species by its proportionally larger eyes and features of the advertisement call. Additionally, genetic distance between the new species and its congeners is 3.0–10.4% based on a fragment of the 16S rRNA gene, which is greater than the threshold typically characterizing distinct species of anurans. Using an integrative approach (molecular, bioacoustics, and adult morphology), we were able to distinguish the new species from other congeneric species. The new species is known only from the type locality where it is threatened by illegal logging and gold mining as well as hydroelectric dams. creator: Diego J. Santana creator: Leandro Alves da Silva creator: Anathielle Caroline Sant’Anna creator: Donald B. Shepard creator: Sarah Mângia uri: https://doi.org/10.7717/peerj.12012 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Santana et al. title: Independent validation of downscaled climate estimates from a coastal Alaska watershed using local historical weather journals link: https://peerj.com/articles/12055 last-modified: 2021-09-10 description: Downscaling coarse global and regional climate models allows researchers to access weather and climate data at finer temporal and spatial resolution, but there remains a need to compare these models with empirical data sources to assess model accuracy. Here, we validate a widely used software for generating North American downscaled climate data, ClimateNA, with a novel empirical data source, 20th century weather journals kept by Admiralty Island, Alaska homesteader, Allen Hasselborg. Using Hasselborg’s journals, we calculated monthly precipitation and monthly mean of the maximum daily air temperature across the years 1926 to 1954 and compared these to ClimateNA data generated from the Hasselborg homestead location and adjacent areas. To demonstrate the utility and potential implications of this validation for other disciplines such as hydrology, we used an established regression equation to generate time series of 95% low duration flow estimates for the month of August using mean annual precipitation from ClimateNA predictions and Hasselborg data. Across 279 months, we found strong correlation between modeled and observed measurements of monthly precipitation (ρ = 0.74) and monthly mean of the maximum daily air temperature (ρ = 0.98). Monthly precipitation residuals (calculated as ClimateNA data - Hasselborg data) generally demonstrated heteroscedasticity around zero, but a negative trend in residual values starting during the last decade of observations may have been due to a shift to the cold-phase Pacific Decadal Oscillation. Air temperature residuals demonstrated a consistent but small positive bias, with ClimateNA tending to overestimate air temperature relative to Hasselborg’s journals. The degree of correlation between weather patterns observed at the Hasselborg homestead site and ClimateNA data extracted from spatial grid cells across the region varied by wet and dry climate years. Monthly precipitation from both data sources tended to be more similar across a larger area during wet years (mean ρ across grid cells = 0.73) compared to dry years (mean ρ across grid cells = 0.65). The time series of annual 95% low duration flow estimates for the month of August generated using ClimateNA and Hasselborg data were moderately correlated (ρ = 0.55). Our analysis supports previous research in other regions which also found ClimateNA to be a robust source for past climate data estimates. creator: Emily R. Williamson creator: Christopher J. Sergeant uri: https://doi.org/10.7717/peerj.12055 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Williamson and Sergeant title: Estrogen receptor alpha regulates the expression of adipogenic genes genetically and epigenetically in rat bone marrow-derived mesenchymal stem cells link: https://peerj.com/articles/12071 last-modified: 2021-09-10 description: Regulation of the efficacy of epigenetic modifiers is regarded as an important control mechanism in the determination and differentiation of stem cell fate. Studies are showing that the effect of estrogen is important in the differentiation of mesenchymal stem cells (MSCs) into adipocytes, osteocytes, and chondrocytes. Activation of certain transcription factors and epigenetic modifications in related genes play an active role in the initiation and completion of adipogenic differentiation. Understanding the role of estrogen in diseases such as obesity, which increases with the onset of menopause, will pave the way for more effective use of estrogen as a therapeutic option. Demonstration of the differentiation tendencies of MSCs change in the presence/absence of estrogen, especially the evaluation of reversible epigenetic changes, will provide valuable information for clinical applications. In this study, the effect of estrogen on the expression of genes involved in adipogenic differentiation of MSCs and accompanying epigenetic modifications was investigated. Our results showed that estrogen affects the expression of adipogenesis-related transcription factors such as PPARy, C/EBPα and Adipsin. In addition, after estrogen treatment, increased accumulation of estrogen receptor alpha (ERα) and repressive epigenetic markers such as H3K27me2 and H3K27me3 were observed on the promoter of given transcription factors. By using co-immunoprecipitation experiments we were also able to show that ERα physically interacts with the zeste homolog 2 (EZH2) H3K27 methyltransferase in MSCs. We propose that the increase of H3K27me2 and H3K27me3 markers on adipogenic genes upon estrogen treatment may be mediated by the direct interaction of ERα and EZH2. Taken together, these findings suggest that estrogen has a role as an epigenetic switcher in the regulation of H3K27 methylation leading to suppression of adipogenic differentiation of MSC. creator: Ceylan V. Bitirim creator: Zeynep B. Ozer creator: Kamil C. Akcali uri: https://doi.org/10.7717/peerj.12071 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Bitirim et al. title: Seagrass contribution to blue carbon in a shallow karstic coastal area of the Gulf of Mexico link: https://peerj.com/articles/12109 last-modified: 2021-09-10 description: Seagrass meadows provide multiple ecosystem services, including carbon sequestration. However, seagrass meadows are among the most threatened ecosystems worldwide. Determining the magnitude of the carbon stocks in seagrass meadows at the regional scale allows for the estimation of their global magnitude and identification of their importance in regional environmental mitigation strategies. The objective of the present study was to determine the structure of seagrass meadows in the Los Petenes Biosfera Reserve (LPBR) and evaluate their contributions to sinks of carbon in this system, located in Yucatan, which is considered the region with the largest seagrass extension in Mexico. Analyses of the seagrass meadows were executed following standardized protocols (spectral analysis, and isotope and carbon stock analyses). The LPBR stores an average of 2.2 ± 1.7 Mg C ha−1 in living biomass and 318 ± 215 Mg C ha−1 in sediment (top 1 m), and this carbon stock decreases with water depth. The seagrass community extends 149,613 ha, which represents the largest organic carbon stock (47 Tg C) documented in seagrass meadows in Mexico. Macroalgae and seagrass represent 76% of the organic carbon stored in sediment. If LPBR seagrass meadows are lost due to natural or anthropogenic impacts, 173 Tg CO2eqemissions could be released, which corresponds to the emissions generated by fossil fuel combustion of 27% of the current Mexican population. This information emphasizes the importance of seagrass meadows as a carbon sink in the region and their contribution to climate change mitigation, thus allowing for the implementation of necessary conservation strategies. creator: Tania C. Cota Lucero creator: Jorge A. Herrera-Silveira uri: https://doi.org/10.7717/peerj.12109 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Cota Lucero and Herrera-Silveira title: Carbon and nitrogen metabolism under nitrogen variation affects flavonoid accumulation in the leaves of Coreopsis tinctoria link: https://peerj.com/articles/12152 last-modified: 2021-09-10 description: Flavonoids are phytochemicals present in medicinal plants and contribute to human health. Coreopsis tinctoria, a species rich in flavonoids, has long been used in traditional medicine and as a food resource. N (nitrogen) fertilization can reduce flavonoid accumulation in C. tinctoria. However, there is limited knowledge regarding N regulatory mechanisms. The aim of this study was to determine the effect of N availability on flavonoid biosynthesis in C. tinctoria and to investigate the relationship between C (carbon) and N metabolism coupled with flavonoid synthesis under controlled conditions. C. tinctoria seedlings were grown hydroponically under five different N levels (0, 0.625, 1.250, 2.500 and 5.000 mM). The related indexes of C, N and flavonoid metabolism of C. tinctoria under N variation were measured and analysed. N availability (low and moderate N levels) regulates enzyme activities related to C and N metabolism, promotes the accumulation of carbohydrates, reduces N metabolite levels, and enhances the internal C/N balance. The flavonoid content in roots and stalks remained relatively stable, while that in leaves peaked at low or intermediate N levels. Flavonoids are closely related to phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate: coenzyme A ligase (4CL), and chalcone-thioase (CHS) activity, significantly positively correlated with carbohydrates and negatively correlated with N metabolites. Thus, C and N metabolism can not only control the distribution of C in amino acid and carbohydrate biosynthesis pathways but also change the distribution in flavonoid biosynthesis pathways, which also provides meaningful information for maintaining high yields while ensuring the nutritional value of crop plants. creator: Zhiyuan Li creator: Hong Jiang creator: Huizhuan Yan creator: Xiumei Jiang creator: Yan Ma creator: Yong Qin uri: https://doi.org/10.7717/peerj.12152 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Li et al. title: Acoustic individuality in the hissing calls of the male black grouse (Lyrurus tetrix) link: https://peerj.com/articles/11837 last-modified: 2021-09-09 description: Acoustic individuality may well play a big role during the mating season of many birds. Black grouse (Lyrurus tetrix) produces two different long-distance calls during mating on leks: rookooing and hissing calls. The first one represents low frequency series of bubbling sounds and the second one represents hissing sound. This hissing represents a signal not produced by the syrinx. We analyzed 426 hissing calls from 24 individuals in Finland and Scotland. We conducted cross-validated discrimination analyses (DFA). The discrimination model classified each call with almost 78% accuracy (conventional result) and the validated DFA revealed 71% output, that is much higher than classification by chance (4%). The most important variables were Frequency 95%, 1st Quartile Frequency, Aggregate Entropy and Duration 90%. We also tested whether between individual variation is higher than within individual variation using PIC (Potential for individual coding) and we found that all acoustic parameters had PIC > 1. We confirmed that hissing call of black grouse is individually distinct. In comparison to the signals produced by the syrinx, non-vocal sounds have been studied rarely and according to our knowledge, this is the second evidence of vocal individuality in avian hissing sounds which are not produced by syrinx. Individuality in the vocalization of the male black grouse may aid females in mating partner selection, and for males it may enable competitor recognition and assessment. Individually distinct hissing calls could be of possible use to monitor individuals on leks. Such a method could overcome problems during traditional monitoring methods of this species, when one individual can be counted multiple times, because catching and traditional marking is problematic in this species. creator: Lucie Hambálková creator: Richard Policht creator: Jiří Horák creator: Vlastimil Hart uri: https://doi.org/10.7717/peerj.11837 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Hambálková et al. title: The importance of molecular characters when morphological variability hinders diagnosability: systematics of the moon jellyfish genus Aurelia (Cnidaria: Scyphozoa) link: https://peerj.com/articles/11954 last-modified: 2021-09-09 description: Cryptic species have been detected across Metazoa, and while no apparent morphological features distinguish them, it should not impede taxonomists from formal descriptions. We accepted this challenge for the jellyfish genus Aurelia, which has a long and confusing taxonomic history. We demonstrate that morphological variability in Aurelia medusae overlaps across very distant geographic localities. Even though some morphological features seem responsible for most of the variation, regional geographic patterns of dissimilarities are lacking. This is further emphasized by morphological differences found when comparing lab-cultured Aurelia coerulea medusae with the diagnostic features in its recent redescription. Previous studies have also highlighted the difficulties in distinguishing Aurelia polyps and ephyrae, and their morphological plasticity. Therefore, mostly based on genetic data, we recognize 28 species of Aurelia, of which seven were already described, 10 are formally described herein, four are resurrected and seven remain undescribed. We present diagnostic genetic characters for all species and designate type materials for newly described and some resurrected species. Recognizing moon jellyfish diversity with formal names is vital for conservation efforts and other studies. This work clarifies the practical implications of molecular genetic data as diagnostic characters, and sheds light on the patterns and processes that generate crypsis. creator: Jonathan W. Lawley creator: Edgar Gamero-Mora creator: Maximiliano M. Maronna creator: Luciano M. Chiaverano creator: Sérgio N. Stampar creator: Russell R. Hopcroft creator: Allen G. Collins creator: André C. Morandini uri: https://doi.org/10.7717/peerj.11954 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Lawley et al. title: Cbl upregulates cysH for hydrogen sulfide production in Aeromonas veronii link: https://peerj.com/articles/12058 last-modified: 2021-09-09 description: Endogenous hydrogen sulfide (H2S) is generated in many metabolism pathways, and has been recognized as a second messenger against antibiotics and reactive oxygen species (ROS). In Aeromonas veronii, Small Protein B (SmpB) plays an important role in resisting stress. The absence of smpB could trigger sulfate assimilation pathway to adapt the nutrient deficiency, of which was mediated by up-regulation of cbl and cys genes and followed with enhancing H2S production. To figure out the mutual regulations of cbl and cys genes, a series of experiments were performed. Compared with the wild type, cysH was down-regulated significantly in cbl deletion by qRT-PCR. The fluorescence analysis further manifested that Cbl had a positive regulatory effect on the promoter of cysJIH. Bacterial one-hybrid analysis and electrophoretic mobility shift assay (EMSA) verified that Cbl bound with the promoter of cysJIH. Collectively, the tolerance to adversity could be maintained by the production of H2S when SmpB was malfunctioned, of which the activity of cysJIH promoter was positively regulated by upstream Cbl protein. The outcomes also suggested the enormous potentials of Aeromonas veronii in environmental adaptability. creator: Yidong Zhang creator: Zebin Liu creator: Yanqiong Tang creator: Xiang Ma creator: Hongqian Tang creator: Hong Li creator: Zhu Liu uri: https://doi.org/10.7717/peerj.12058 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Zhang et al. title: Apis andreniformis associated Actinomycetes show antimicrobial activity against black rot pathogen (Xanthomonas campestris pv. campestris) link: https://peerj.com/articles/12097 last-modified: 2021-09-09 description: This study aimed to investigate cultivable actinomycetes associated with rare honey bee species in Thailand and their antagonistic activity against plant pathogenic bacteria. Actinomycetes were selectively isolated from the black dwarf honey bee (Apis andreniformis). A total of 64 actinomycete isolates were obtained with Streptomyces as the predominant genus (84.4%) followed by Micromonospora (7.8%), Nonomuraea (4.7%) and Actinomadura (3.1%). All isolates were screened for antimicrobial activity against Xanthomonas campestris pv. campestris, Pectobacterium carotovorum and Pseudomonas syringae pv. sesame. Three isolates inhibited the growth of X. campestris pv. campestris during in vitro screening. The crude extracts of two isolates (ASC3-2 and ASC5-7P) had a minimum inhibitory concentration (MIC) of 128 mg L−1against X. campestris pv. campestris. For isolate ACZ2-27, its crude extract showed stronger inhibitory effect with a lower MIC value of 64 mg L−1 against X. campestris pv. campestris. These three active isolates were identified as members of the genus Streptomyces based on their 16S rRNA gene sequences. Phylogenetic analysis based on the maximum likelihood algorithm showed that isolate ACZ2-27, ASC3-2 and ASC5-7P were closely related to Streptomyces misionensis NBRC 13063T (99.71%), Streptomyces cacaoi subsp. cacaoi NBRC 12748T (100%) and Streptomyces puniceus NBRC 12811T (100%), respectively. In addition, representative isolates from non-Streptomyces groups were identified by 16S rRNA gene sequence analysis. High similarities were found with members of the genera Actinomadura, Micromonospora and Nonomuraea. Our study provides evidence of actinomycetes associated with the black dwarf honey bee including members of rare genera. Antimicrobial potential of these insect associated Streptomyces was also demonstrated especially the antibacterial activity against phytopathogenic bacteria. creator: Yaowanoot Promnuan creator: Saran Promsai creator: Wasu Pathom-aree creator: Sujinan Meelai uri: https://doi.org/10.7717/peerj.12097 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Promnuan et al. title: Tackling the phylogenetic conundrum of Hydroidolina (Cnidaria: Medusozoa: Hydrozoa) by assessing competing tree topologies with targeted high-throughput sequencing link: https://peerj.com/articles/12104 last-modified: 2021-09-09 description: Higher-level relationships of the Hydrozoan subclass Hydroidolina, which encompasses the vast majority of medusozoan cnidarian species diversity, have been elusive to confidently infer. The most widely adopted phylogenetic framework for Hydroidolina based on ribosomal RNA data received low support for several higher level relationships. To address this issue, we developed a set of RNA baits to target more than a hundred loci from the genomes of a broad taxonomic sample of Hydroidolina for high-throughput sequencing. Using these data, we inferred the relationships of Hydroidolina using maximum likelihood and Bayesian approaches. Both inference methods yielded well-supported phylogenetic hypotheses that largely agree with each other. Using maximum likelihood and Baysian hypothesis testing frameworks, we found that several alternate topological hypotheses proposed previously may be rejected in light of the genomic data generated for this study. Both the maximum likelihood and Bayesian topologies inferred herein consistently score well across testing frameworks, suggesting that their consensus represents the most likely phylogenetic hypothesis of Hydroidolina. This phylogenetic framework places Aplanulata as sister lineage to the remainder of Hydroidolina. This is a strong deviation from previous phylogenetic analyses that placed Capitata or Siphonophorae as sister group to the remainder of Hydroidolina. Considering that Aplanulata represents a lineage comprised of species that for the most part possess a life cycle involving a solitary polyp and free-swimming medusa stage, the phylogenetic hypotheses presented herein have potentially large implications for clarifying the evolution of life cycles, coloniality, and the division of labor in Hydrozoa as taxon sampling for phylogenetic analyses becomes more complete. creator: Bastian Bentlage creator: Allen G. Collins uri: https://doi.org/10.7717/peerj.12104 license: https://creativecommons.org/publicdomain/zero/1.0/ rights: ©2021 Bentlage and Collins title: An artificial neural network for automated behavioral state classification in rats link: https://peerj.com/articles/12127 last-modified: 2021-09-09 description: Accurate behavioral state classification is critical for many research applications. Researchers typically rely upon manual identification of behavioral state through visual inspection of electrophysiological signals, but this approach is time intensive and subject to low inter-rater reliability. To overcome these limitations, a diverse set of algorithmic approaches have been put forth to automate the classification process. Recently, novel machine learning approaches have been detailed that produce rapid and highly accurate classifications. These approaches however, are often computationally expensive, require significant expertise to implement, and/or require proprietary software that limits broader adoption. Here we detail a novel artificial neural network that uses electrophysiological features to automatically classify behavioral state in rats with high accuracy, sensitivity, and specificity. Common parameters of interest to sleep scientists, including state-dependent power spectra and homeostatic non-REM slow wave activity, did not significantly differ when using this automated classifier as compared to manual scoring. Flexible options enable researchers to further increase classification accuracy through manual rescoring of a small subset of time intervals with low model prediction certainty or further decrease researcher time by generalizing trained networks across multiple recording days. The algorithm is fully open-source and coded within a popular, and freely available, software platform to increase access to this research tool and provide additional flexibility for future researchers. In sum, we have developed a readily implementable, efficient, and effective approach for automated behavioral state classification in rats. creator: Jacob G. Ellen creator: Michael B. Dash uri: https://doi.org/10.7717/peerj.12127 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Ellen and Dash title: Plastic response of Medicago sativa L. root system traits and cold resistance to simulated rainfall events link: https://peerj.com/articles/11962 last-modified: 2021-09-09 description: Climate change (rainfall events and global warming) affects the survival of alfalfa (Medicago sativa L.) in winter. Appropriate water management can quickly reduce the mortality of alfalfa during winter. To determine how changes in water affect the cold resistance of alfalfa, we explored the root system traits under different rainfall events and the effects on cold resistance in three alfalfa cultivars. These were exposed to three simulated rainfall events (SRE) × two phases in a randomized complete block design with six replications. The three cultivars were WL168, WL353 and WL440, and the three SRE were irrigation once every second day (D2), every four days (D4) and every eight days (D8). There were two phases: before cold acclimation and after cold acclimation. Our results demonstrated that a period of exposure to low temperature was required for alfalfa to achieve maximum cold resistance. The root system tended toward herringbone branching under D8, compared with D2 and D4, and demonstrated greater root biomass, crown diameter, root volume, average link length and topological index. Nevertheless, D8 had less lateral root length, root surface area, specific root length, root forks and fractal dimensions. Greater root biomass and topological index were beneficial to cold resistance in alfalfa, while more lateral roots and root forks inhibited its ability to survive winter. Alfalfa roots had higher proline, soluble sugar and starch content in D8 than in D2 and D4. In contrast, there was lower malondialdehyde in D8, indicating that alfalfa had better cold resistance following a longer irrigation interval before winter. After examining root biomass, root system traits and physiological indexes we concluded that WL168 exhibited stronger cold resistance. Our results contribute to greater understanding of root and cold stress, consequently providing references for selection of cultivars and field water management to improve cold resistance of alfalfa in the context of changes in rainfall patterns. creator: Zhensong Li creator: Liqiang Wan creator: Shuo Li creator: Xianglin Li creator: Feng He creator: Zongyong Tong uri: https://doi.org/10.7717/peerj.11962 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Li et al. title: Risk assessment of freezing injury during overwintering of wheat in the northern boundary of the Winter Wheat Region in China link: https://peerj.com/articles/12154 last-modified: 2021-09-09 description: Freezing injury is one of the main restriction factors for winter wheat production, especially in the northern part of the Winter Wheat Region in China. It is very important to assess the risk of winter wheat-freezing injury. However, most of the existing climate models are complex and cannot be widely used. In this study, Zunhua which is located in the northern boundary of Winter Wheat Region in China is selected as research region, based on the winter meteorological data of Zunhua from 1956 to 2016, seven freezing disaster-causing factors related to freezing injury were extracted to formulated the freezing injury index (FII) of wheat. Referring to the historical wheat-freezing injury in Zunhua and combining with the cold resistance identification data of the National Winter Wheat Variety Regional Test (NWWVRT), consistency between the FII and the actual freezing injury situation was tested. Furthermore, the occurrence law of freezing injury in Zunhua during the past 60 years was analyzed by Morlet wavelet analyze, and the risk of freezing injury in the short term was evaluated. Results showed that the FII can reflect the occurrence of winter wheat-freezing injury in Zunhua to a certain extent and had a significant linear correlation with the dead tiller rate of wheat (P = 0.014). The interannual variation of the FII in Zunhua also showed a significant downward trend (R2 = 0.7412). There are two cycles of freezing injury in 60 years, and it showed that there’s still exist a high risk in the short term. This study provides reference information for the rational use of meteorological data for winter wheat-freezing injury risk assessment. creator: Jiahui Guo creator: Xionghui Bai creator: Weiping Shi creator: Ruijie Li creator: Xingyu Hao creator: Hongfu Wang creator: Zhiqiang Gao creator: Jie Guo creator: Wen Lin uri: https://doi.org/10.7717/peerj.12154 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Guo et al. title: CBF transcription factors involved in the cold response of Camellia japonica (Naidong) link: https://peerj.com/articles/12155 last-modified: 2021-09-09 description: CBFs belong to the ERF subfamily of the AP2 supergene family and often play an important role in the cold acclimation of temperate plants. However, the role of CBFs in Camellia japonica (Naidong), the only Camellia japonica population found in the temperate zones of China, remains unclear. It is very important to study the genetic composition of C. japonica (Naidong) to adapt to low temperature for Camellia species. Using full-length transcriptome data, we identified four CjCBF genes that respond to cold stress and analyzed their evolutionary relationships, domains, and expression patterns. The phylogeny of CBFs of 19 angiosperms divided the genes into three categories, and the four CjCBFs belong to a small subcluster. The strong response of CjCBF1 to cold treatment and its sustained high level of expression indicated that it plays an important role in the process of cold acclimation. A yeast two-hybrid assay revealed an interaction between CjCBF1, CjCBF2, and CjCBF5, and subcellular localization confirmed this finding. The expression of CjCBFs was tissue-specific: CBF1 was mainly expressed in leaves, and CBF3 was mainly expressed in stem. The responses of the four CjCBFs to drought and high temperature and the effect of light were also characterized. Our study provides new insight into the role of CBFs in the cold response in C. japonica (Naidong). creator: Menglong Fan creator: Rui Zhou creator: Qinghua Liu creator: Yingkun Sun uri: https://doi.org/10.7717/peerj.12155 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Fan et al. title: High bycatch rates of manta and devil rays in the “small-scale” artisanal fisheries of Sri Lanka link: https://peerj.com/articles/11994 last-modified: 2021-09-08 description: BackgroundExpanding fisheries in developing nations like Sri Lanka have a significant impact on threatened marine species such as elasmobranchs. Manta and devil (mobulid) rays have some of the most conservative life history strategies of any elasmobranch, and even low to moderate levels of bycatch from gillnet fisheries may lead to significant population declines. A lack of information on life history, demographics, population trends, and fisheries impacts hinders effective management measures for these species.MethodWe report on mobulid fishery landings over nine years between 2011 and 2020 across 38 landing sites in Sri Lanka. We collected data on catch numbers, body sizes, sex, and maturity status for five mobulid species. We used a Bayesian state-space model to estimate monthly country-wide catch rates and total annual landings of mobulid rays. We used catch curve analyses to estimate total mortality for Mobula mobular, and evaluated trends in recorded body sizes across the study period for M. mobular, M. birostris, M. tarapacana and M. thurstoni.ResultsWe find that catch rates have declined an order of magnitude for all species across the study period, and that total annual captures of mobulid rays by the Sri Lankan artisanal fishing fleet exceed the estimated annual captures of mobulids in all global, industrial purse seine fisheries combined. Catch curve analyses suggest that M. mobular is being fished at rates far above the species’ intrinsic population growth rate, and the average sizes of all mobulids in the fishery except for M. birostris are declining. Collectively, these findings suggest overfishing of mobulid ray populations in the northern Indian Ocean by Sri Lankan artisanal fisheries. We recommend strengthening the management of these species through improved implementation of CITES, CMS, and regional fisheries management actions. In addition, we report on the demographic characteristics of mobulids landed in Sri Lanka and provide the first record of M. eregoodoo in the country. creator: Daniel Fernando creator: Joshua D. Stewart uri: https://doi.org/10.7717/peerj.11994 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Fernando and Stewart title: Inter-element variation in the bone histology of Anteosaurus (Dinocephalia, Anteosauridae) from the Tapinocephalus Assemblage Zone of the Karoo Basin of South Africa link: https://peerj.com/articles/12082 last-modified: 2021-09-08 description: Despite its abundance in the Permian fossil record of South Africa, little is known about the life history of Anteosaurus. Here we examine the bone microstructure of multiple skeletal elements of Anteosaurus from the Tapinocephalus Assemblage Zone of the Karoo Basin. The bone histology of Anteosaurus magnificus reveals that the cortex is composed of highly vascularized, uninterrupted fibrolamellar bone tissue surrounding the inner spongy medullary region. However, the histology of two ribs and a previously described femur of another Anteosaurus taxon revealed an interrupted growth pattern with lines of arrested growth and peripheral rest lines occurring in the compacta, indicating periodic pauses in growth possibly linked to the slowing down of growth during maturity. Given that the fibula of the same individual has well-vascularised fibrolamellar bone tissue without any growth marks in the cortex; this suggests variation in skeletal growth. Based on our histological results, three growth dynamic stages are deduced for the genus Anteosaurus: (i) the earliest growth stage is represented by the predominance of highly vascularized, uninterrupted fibrolamellar bone tissue in the inner cortex, which suggests rapid periosteal bone deposition during early ontogeny; (ii) the next stage of growth shows periodic interruptions in the bone deposition as indicated by the deposition of lines of arrested growth; (iii) the third stage shows the development of lamellar bone tissue with rest lines in the peripheral part of the cortex suggesting a slowing down of growth prior to death. Most of the skeletal elements are characterized by thick bone walls, extensive secondary reconstruction and the complete infilling of the medullary cavity. However, the radius and a previously studied femur have open medullary cavities with struts of bony trabeculae. Based on histologic structures and comparisons with extant taxa, it is likely that Anteosaurus may have been more terrestrial as its osteology point towards terrestriality, but it may have occasionally inhabited ephemeral pools like modern semi-aquatic Hippopotamus. creator: Mohd Shafi Bhat creator: Christen D. Shelton creator: Anusuya Chinsamy uri: https://doi.org/10.7717/peerj.12082 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Bhat et al. title: No adverse effect of a maternal high carbohydrate diet on their offspring, in rainbow trout (Oncorhynchus mykiss) link: https://peerj.com/articles/12102 last-modified: 2021-09-08 description: In order to develop a sustainable salmonid aquaculture, it is essential to continue to reduce the use of the protein-rich fishmeal. One promising solution to do so is the use of plant-derived carbohydrates in diet destined to broodstock. However, in mammals, the reduction of protein content (replaced by carbohydrates) in parental diet is known to have strong adverse effects on offspring phenotypes and metabolism. For the first time, the effect of a paternal and a maternal high carbohydrate-low protein diet was assessed on progeny at long term in the rainbow trout. A 30% protein diminution in both males and females broodstock diet during 10 month and 5 months, respectively, did not trigger adverse consequences on their offspring. At the molecular level, offspring transcriptomes were not significantly altered, emphasizing no effect on metabolism. Tenuous differences in the biochemical composition of the liver and the viscera were observed. The recorded effects remained in the normal range of value and accordingly offspring growth were not negatively affected over the long term. Overall, we demonstrated here that a 30% protein diminution during gametogenesis is feasible, confirming the possibility to increase the proportion of plant-derived carbohydrates in female broodstock diets to replace fishmeal proteins. creator: Therese Callet creator: Hongyan Li creator: Anne Surget creator: Frederic Terrier creator: Franck Sandres creator: Anthony Lanuque creator: Stephane Panserat creator: Lucie Marandel uri: https://doi.org/10.7717/peerj.12102 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Callet et al. title: High throughput sequencing-based analysis of the soil bacterial community structure and functions of Tamarix shrubs in the lower reaches of the Tarim River link: https://peerj.com/articles/12105 last-modified: 2021-09-08 description: Tamarix is a dominant species in the Tarim River Basin, the longest inland river in China. Tamarix plays an important role in the ecological restoration of this region. In this study, to investigate the soil bacterial community diversity in Tamarix shrubs, we collected soil samples from the inside and edge of the canopy and the edge of nebkhas and non-nebkhas Tamarix shrubs located near the Yingsu section in the lower reaches of Tarim River. High throughput sequencing technology was employed to discern the composition and function of soil bacterial communities in nebkhas and non-nebkhas Tamarix shrubs. Besides, the physicochemical properties of soil and the spatial distribution characteristics of soil bacteria and their correlation were analyzed. The outcomes of this analysis demonstrated that different parts of Tamarix shrubs had significantly different effects on soil pH, total K (TK), available K (AK), ammonium N (NH4+), and available P (AP) values (P < 0.05), but not on soil moisture (SWC), total salt (TDS), electrical conductivity (EC), organic matter (OM), total N (TN), total P (TP), and nitrate N (NO3−) values. The soil bacterial communities identified in Tamarix shrubs were categorized into two kingdoms, 71 phyla, 161 classes, 345 orders, 473 families, and 702 genera. Halobacterota, unidentified bacteria, and Proteobacteria were found to be dominant phyla. The correlation between the soil physicochemical factors and soil bacterial community was analyzed, and as per the outcomes OM, AK, AP, EC, and NH4+ were found to primarily affect the structure of the soil bacterial community. SWC, TK and pH were positively correlated with each other, but negatively correlated with other soil factors. At the phyla level, a significantly positive correlation was observed between the Halobacterota and AP, OM as well as Bacteroidota and AK (P < 0.01), but a significantly negative correlation was observed between the Chloroflexi and AK, EC (P < 0.01). The PICRUSt software was employed to predict the functional genes. A total of 6,195 KEGG ortholog genes were obtained. The function of soil bacteria was annotated, and six metabolic pathways in level 1, 41 metabolic pathways in level 2, and 307 metabolic pathways in level 3 were enriched, among which the functional gene related to metabolism, genetic information processing, and environmental information processing was found to have the dominant advantage. The results showed that the nebkhas and canopy of Tamarix shrubs had a certain enrichment effect on soil nutrients content, and bacterial abundance and significant effects on the structure and function of the soil bacterial community. creator: Fangnan Xiao creator: Yuanyuan Li creator: Guifang Li creator: Yaling He creator: Xinhua Lv creator: Li Zhuang creator: Xiaozhen Pu uri: https://doi.org/10.7717/peerj.12105 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Xiao et al. title: Whole-genome comparative analysis at the lineage/sublineage level discloses relationships between Mycobacterium tuberculosis genotype and clinical phenotype link: https://peerj.com/articles/12128 last-modified: 2021-09-08 description: BackgroundHuman tuberculosis (TB) caused by members of the Mycobacterium tuberculosis complex (MTBC) is the main cause of death among infectious diseases worldwide. Pulmonary TB (PTB) is the most common clinical phenotype of the disease, but some patients develop an extrapulmonary (EPTB) phenotype in which any organ or tissue can be affected. MTBC species include nine phylogenetic lineages, with some appearing globally and others being geographically restricted. EPTB can or not have pulmonary involvement, challenging its diagnosis when lungs are not implicated, thus causing an inadequate treatment. Finding evidence of a specific M. tuberculosis genetic background associated with EPTB is epidemiologically relevant due to the virulent and multidrug-resistant strains isolated from such cases. Until now, the studies conducted to establish associations between M. tuberculosis lineages and PTB/EPTB phenotypes have shown inconsistent results, which are attributed to the strain predominance from specific M. tuberculosis lineages/sublineages in the samples analyzed and the use of low-resolution phylogenetic tools that have impaired sublineage discrimination abilities. The present work elucidates the relationships between the MTBC strain lineages/sublineages and the clinical phenotypes of the disease as well as the antibiotic resistance of the strains.MethodsTo avoid biases, we retrieved the raw genomic reads (RGRs) of all (n = 245) the M. tuberculosis strains worldwide causing EPTB available in databases and an equally representative sample of the RGRs (n = 245) of PTB strains. A multiple alignment was constructed, and a robust maximum likelihood phylogeny based on single-nucleotide polymorphisms was generated, allowing effective strain lineage/sublineage assignment.ResultsA significant Odds Ratio (OR range: 1.8–8.1) association was found between EPTB and the 1.1.1, 1.2.1, 4.1.2.1 and ancestral Beijing sublineages. Additionally, a significant association between PTB with 4.3.1, 4.3.3, and 4.5 and Asian African 2 and Europe/Russia B0/W148 modern Beijing sublineages was found. We also observed a significant association of Lineage 3 strains with multidrug resistance (OR 3.8; 95% CI [1.1–13.6]), as well as between modern Beijing sublineages and antibiotic resistance (OR 4.3; 3.8–8.6). In this work, it was found that intralineage diversity can drive differences in the immune response that triggers the PTB/EPTB phenotype. creator: Andrea Monserrat Negrete-Paz creator: Gerardo Vázquez-Marrufo creator: Ma. Soledad Vázquez-Garcidueñas uri: https://doi.org/10.7717/peerj.12128 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Negrete-Paz et al. title: Apis mellifera propolis enhances apoptosis and invasion inhibition in head and neck cancer cells link: https://peerj.com/articles/12139 last-modified: 2021-09-08 description: BackgroundPropolis is a resinous product accumulated from several plant sources that possess a wide range of therapeutic properties, including anti-cancer activities. However, the role of honeybee-produced propolis on head and neck squamous carcinoma (HNSCC) is not well understood. The aim of this study was to investigate the effects of Apis mellifera propolis on apoptosis and invasiveness in HNSCC cell lines.MethodsEthyl acetate extract of propolis (EAEP) was prepared from A. mellifera beehives using liquid–liquid extraction. High-performance liquid chromatography coupled with electrospray ionization-time of flight-mass spectrometry (HPLC-ESI-TOF-MS) was used to determine the flavonoids in EAEP. Isogenic HNSCC cell lines derived from primary (HN30 and HN4) and metastatic site (HN31 and HN12) were used in this study. The cytotoxicity, apoptosis, invasion, and MMP activity of EAEP on HNSCC cells were determined using an MTT assay, flow cytometry, Matrigel invasion assay, and gelatinase zymography, respectively.ResultsWe found that EAEP exhibited cytotoxic activity and induced apoptosis in the HNSCC cell lines. Furthermore, EAEP significantly decreased HNSCC cell invasion by reducing MMP-2 and MMP-9 activity. Two flavonoids, galangin and apigenin, were identified in EAEP by HPLC-ESI-TOF-MS. The results suggest that EAEP promotes apoptosis and exerts anti-invasion potential by inhibiting MMP-2 and MMP-9 activity in HNSCC cell lines. These inhibitory effects may be mediated by galangin and apigenin. creator: Nattisa Niyomtham creator: Sittichai Koontongkaew creator: Boon-ek Yingyongnarongkul creator: Kusumawadee Utispan uri: https://doi.org/10.7717/peerj.12139 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Niyomtham et al. title: Dendrobaena veneta avoids ethyl pentanoate and ethyl hexanoate, two compounds produced by the soil fungus Geotrichum candidum link: https://peerj.com/articles/12148 last-modified: 2021-09-08 description: Earthworms shape the biological and physicochemical qualities of the soil they choose to reside in, but our understanding of the specific chemicals that attract or repel a particular species of earthworm remains incomplete. Current research indicates that some species feed on and are attracted to fungi, such as Geotrichum candidum. In the present study, as part of our continuing effort to characterize mechanisms of earthworm chemosensation, we tested whether ethyl hexanoate and ethyl pentanoate, two compounds produced by G. candidum, are appetitive to the European nightcrawler (Dendrobaena veneta). In a soil T-maze, both of these compounds significantly repelled individual earthworms in a dosage-dependent manner, this result ran counter to our initial hypothesis. D. veneta also avoided ethyl hexanoate and ethyl pentanoate in an assay we specifically developed to test an earthworms aversion to chemical stimuli in soil. In both of these assays, ethyl hexanoate was aversive at lower concentrations than ethyl pentanoate. These findings further clarify our understanding of the chemical cues that trigger the decision of D. veneta to select a particular soil-environment, and emphasize that different earthworm species may react very differently to commonly encountered chemical stimuli. creator: Eileen M.S. Reed creator: Mariel O. O’Connor creator: Ione C. Johnson creator: Wayne L. Silver creator: Cecil J. Saunders uri: https://doi.org/10.7717/peerj.12148 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Reed et al. title: Probabilistically sampled and spectrally clustered plant species using phenotypic characteristics link: https://peerj.com/articles/11927 last-modified: 2021-09-07 description: Phenotypic characteristics of a plant species refers to its physical properties as cataloged by plant biologists at different research centers around the world. Clustering species based upon their phenotypic characteristics is used to obtain diverse sets of parents that are useful in their breeding programs. The Hierarchical Clustering (HC) algorithm is the current standard in clustering of phenotypic data. This algorithm suffers from low accuracy and high computational complexity issues. To address the accuracy challenge, we propose the use of Spectral Clustering (SC) algorithm. To make the algorithm computationally cheap, we propose using sampling, specifically, Pivotal Sampling that is probability based. Since application of samplings to phenotypic data has not been explored much, for effective comparison, another sampling technique called Vector Quantization (VQ) is adapted for this data as well. VQ has recently generated promising results for genotypic data. The novelty of our SC with Pivotal Sampling algorithm is in constructing the crucial similarity matrix for the clustering algorithm and defining probabilities for the sampling technique. Although our algorithm can be applied to any plant species, we tested it on the phenotypic data obtained from about 2,400 Soybean species. SC with Pivotal Sampling achieves substantially more accuracy (in terms of Silhouette Values) than all the other proposed competitive clustering with sampling algorithms (i.e. SC with VQ, HC with Pivotal Sampling, and HC with VQ). The complexities of our SC with Pivotal Sampling algorithm and these three variants are almost the same because of the involved sampling. In addition to this, SC with Pivotal Sampling outperforms the standard HC algorithm in both accuracy and computational complexity. We experimentally show that we are up to 45% more accurate than HC in terms of clustering accuracy. The computational complexity of our algorithm is more than a magnitude less than that of HC. creator: Aditya A. Shastri creator: Kapil Ahuja creator: Milind B. Ratnaparkhe creator: Yann Busnel uri: https://doi.org/10.7717/peerj.11927 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Shastri et al. title: Potential applications of personality assessments to the management of non-human primates: a review of 10 years of study link: https://peerj.com/articles/12044 last-modified: 2021-09-07 description: Studies of primate personality have become increasingly common over the past three decades. Recently, studies have begun to focus on the health, welfare and conservation implications of personality, and the potential applications of incorporating quantitative personality assessments into animal management programmes. However, this literature is dispersed across a multitude of settings and scientific disciplines. We conducted a review of nonhuman primate personality studies relevant to these issues published since 2010, following on from an earlier review. The databases ScienceDirect, PubMed and Web of Science were used to identify relevant articles. After eliminating irrelevant or duplicate papers, 69 studies were selected. Our review reveals that, while primate personality research is carried out on a range of species, there is strong taxonomic bias. While 28 species appeared within the reviewed literature, 52% of studies were carried out on just five species. Further, the most common research focus (43%) was validating new assessment methods or describing personality in different species, rather than exploring the links between personality and animal welfare using existing validated methods. However, among the remaining studies that did explore the role of animal personality in husbandry, health, and welfare, we identified progression towards integrating personality data into various aspects of animal management. Evidence suggests the assessment of personality may benefit social group management, enrichment practices, training protocols, health and welfare monitoring, and conservation planning for endangered species. We argue that further research which develops our understanding of primate personality and its influence in these areas will provide a valuable tool to inform animal management practices. creator: Max Norman creator: Lewis J. Rowden creator: Guy Cowlishaw uri: https://doi.org/10.7717/peerj.12044 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Norman et al. title: Interaction of interregional O3 pollution using complex network analysis link: https://peerj.com/articles/12095 last-modified: 2021-09-07 description: In order to improve the accuracy of air pollution management and promote the efficiency of coordinated inter-regional prevention and control, this study analyzes the interaction of O3 in Qilihe District, Lanzhou City, China. Data used for analysis was obtained from 63 air quality monitoring stations between November 2017 and October 2018. This paper uses complex network theory to describe the network structure characteristics of O3 pollution spatial correlation. On this basis, the node importance method is used to mine the sub-network with the highest spatial correlation in the O3 network, and use transfer entropy theory to analyse the interaction of pollutants between regions. The results show that the O3 area of Qilihe District, Lanzhou City can be divided into three parts: the urban street community type areas in urban areas, the township and village type areas in mountain areas and the scattered areas represented by isolated nodes. An analysis of the mutual influence of O3 between each area revealed that the impact of O3 on each monitoring station in adjacent areas will vary considerably. Therefore these areas cannot be governed as a whole, and the traditional extensive management measures based on administrative divisions cannot be used to replace all other regional governance measures. There is the need to develop a joint prevention and control mechanism tailored to local conditions in order to improve the accuracy and efficiency of O3 governance. creator: Qiang Zhang creator: Yunan Zhu creator: Dianxiang Xu creator: Jiaqiong Yuan creator: Zhihe Wang creator: Yong Li creator: Xueyan Liu uri: https://doi.org/10.7717/peerj.12095 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Zhang et al. title: Identification of immune related cells and crucial genes in the peripheral blood of ankylosing spondylitis by integrated bioinformatics analysis link: https://peerj.com/articles/12125 last-modified: 2021-09-07 description: BackgroundAnkylosing spondylitis (AS) is a progressive rheumatic disease and studies reveal that the immune system is critical for the pathogenesis of AS. In the present study, various bioinformatics analysis methods were comprehensively applied, designed to identify potential key genes and inflammation states of AS.MethodsThe transcriptome profiles of GSE25101 and GSE73754 obtained from the Gene Expression Omnibus (GEO) database were merged for subsequent analyses. The differentially expressed genes (DEGs) were identified using the Bioconductor package Limma and threshold values. Functional enrichment and pathway enrichment analyses were performed using the clusterProfiler package and Gene Set Enrichment Analysis (GSEA). Next, protein–protein interaction (PPI) network of the identified DEGs was constructed by the online database, the Search Tool for the Retrieval of Interacting Genes (STRING), visualization and analysis were performed through Cytoscape software. Subsequently, we applied CIBERSORT algorithm to identify subpopulation proportions of immune cells in peripheral blood samples. Finally, we validated the hub genes with the GSE18781 dataset. Samples were collected from patients to validate gene and protein expression using qRT-PCR and ELISA.ResultsA total of 334 DEGs were identified, including 182 upregulated and 152 downregulated DEGs, between AS patients and normal human controls, which were primarily involved in immune response, autophagy, and natural killer cell-mediated cytotoxicity. The most prominent module and candidate biomarkers were identified from the PPI network. Biomarkers were selected for validation and their expressions were significantly decreased in peripheral blood samples which was consistent with transcriptome sequencing results. Nine genes with AUC > 0.70 were considered to be AS hub genes for ROC curve analysis, including GZMA, GZMK, PRF1, GNLY, NKG7, KLRB1, KLRD1, IL2RB and CD247. Furthermore, CIBERSORT results suggest that AS contained a higher proportion of CD8+ T cells, naive CD4+ T cells, neutrophils, and lower levels of gamma delta T cells compared with the normal controls.ConclusionIn this study, we identified DEGs combined with their closely related biological functions and propose that granule-associated proteins and immune infiltration maybe involved in the progression of ankylosing spondylitis. These validated hub genes may provide new perspectives for understanding the molecular mechanisms of ankylosing spondylitis. creator: Yang Zheng creator: Bingbing Cai creator: Conglin Ren creator: Haipeng Xu creator: Weibin Du creator: Yijiang Wu creator: Fu Lin creator: Helou Zhang creator: Renfu Quan uri: https://doi.org/10.7717/peerj.12125 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Zheng et al. title: Investigation of multiple sclerosis-related pathways through the integration of genomic and proteomic data link: https://peerj.com/articles/11922 last-modified: 2021-09-06 description: BackgroundMultiple sclerosis (MS) has a complex pathophysiology, variable clinical presentation, and unpredictable prognosis; understanding the underlying mechanisms requires combinatorial approaches that warrant the integration of diverse molecular omics data.MethodsHere, we combined genomic and proteomic data of the same individuals among a Turkish MS patient group to search for biologically important networks. We previously identified differentially-expressed proteins by cerebrospinal fluid proteome analysis of 179 MS patients and 42 non-MS controls. Among this study group, 11 unrelated MS patients and 60 independent, healthy controls were subjected to whole-genome SNP genotyping, and genome-wide associations were assessed. Pathway enrichment analyses of MS-associated SNPs and differentially-expressed proteins were conducted using the functional enrichment tool, PANOGA.ResultsNine shared pathways were detected between the genomic and proteomic datasets after merging and clustering the enriched pathways. Complement and coagulation cascade was the most significantly associated pathway (hsa04610, P = 6.96 × 10−30). Other pathways involved in neurological or immunological mechanisms included adherens junctions (hsa04520, P = 6.64 × 10−25), pathogenic Escherichia coli infection (hsa05130, P = 9.03 × 10−14), prion diseases (hsa05020, P = 5.13 × 10−13).ConclusionWe conclude that integrating multiple datasets of the same patients helps reducing false negative and positive results of genome-wide SNP associations and highlights the most prominent cellular players among the complex pathophysiological mechanisms. creator: Elif Everest creator: Ege Ülgen creator: Ugur Uygunoglu creator: Melih Tutuncu creator: Sabahattin Saip creator: Osman Uğur Sezerman creator: Aksel Siva creator: Eda Tahir Turanli uri: https://doi.org/10.7717/peerj.11922 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Everest et al. title: A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection link: https://peerj.com/articles/11996 last-modified: 2021-09-06 description: The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering. creator: Panjaporn Wongpayak creator: Orapan Meesungnoen creator: Somchai Saejang creator: Pakpoom Subsoontorn uri: https://doi.org/10.7717/peerj.11996 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Wongpayak et al. title: Impact of umbilical cord arterial pH, gestational age, and birth weight on neurodevelopmental outcomes for preterm neonates link: https://peerj.com/articles/12043 last-modified: 2021-09-06 description: BackgroundThe aim of this study was to determine the impact of umbilical cord arterial pH, gestational age, and birth weight on neurodevelopmental outcomes for preterm neonates.MethodsWe examined 112 neonates. Inclusion criteria were: Saturations greater than 88%, and heart rates between 100–205 beats per minute.MeasurementsWe assessed several neurodevelopmental factors as part of the Brazelton Neonatal Behavioral Assessment Scale (NBAS), 4th edition, such as asymmetric tonic neck reflex (ATNR), motor maturity, response to sensory stimuli, habituation, and state regulation. Initial assessment parameters such as APGAR score and umbilical cord arterial pH were used to assess neonates.ResultsWe found a strong correlation between the presence of the sucking reflex and umbilical cord arterial pH (r = 0.32; p = 0.018981). Umbilical cord arterial pH was also correlated with the presence of asymmetric tonic neck reflex (r = 0.27; p = 0.047124), cost of attention (r = 0.31; p = 0.025381) and general motor maturity (r = 0.34; p = 0.011741).ConclusionsWe found that the sucking reflex may be affected in infants with low umbilical cord arterial pH values. Practitioners and parents can use the NBAS to help determine neurodevelopmental factors and outcomes in preterm infants, possibly leading to safer and more effective feeding practices and interventions. creator: Roksana Malak creator: Dorota Sikorska creator: Marta Rosołek creator: Ewa Baum creator: Ewa Mojs creator: Przemysław Daroszewski creator: Monika Matecka creator: Brittany Fechner creator: Włodzimierz Samborski uri: https://doi.org/10.7717/peerj.12043 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Malak et al. title: Downregulation of PPA2 expression correlates with poor prognosis of kidney renal clear cell carcinoma link: https://peerj.com/articles/12086 last-modified: 2021-09-06 description: BackgroundKidney renal clear cell carcinoma (KIRC) is the most common subtype of kidney cancer. Inorganic pyrophosphatase (PPA2) is an enzyme that catalyzes the hydrolysis of pyrophosphate to inorganic phosphate; few studies have reported its significance in cancers. Therefore, we aimed to explore the prognostic value of PPA2 in KIRC.MethodsPPA2 expression was detected via immunohistochemistry in a tissue chip containing specimens from 150 patients with KIRC. We evaluated the correlation between PPA2 expression, clinicopathological characteristics, and survival. Data from online databases and another cohort (paraffin-embedded specimens from 10 patients with KIRC) were used for external validation.ResultsPPA2 expression was significantly lower in KIRC tissues than in normal renal tissues (p < 0.0001). Low expression of PPA2 was significantly associated with a high histologic grade and poor prognosis. The differential expression of PPA2 was validated at the gene and protein levels. Multivariate Cox regression analysis showed that PPA2 expression was an independent prognostic factor in patients with KIRC. Gene set enrichment analysis suggested that decreased expression of PPA2 might be related to the epithelial-mesenchymal transition in KIRC.ConclusionsOur study demonstrated that PPA2 is an important energy metabolism-associated biomarker correlated with a favorable prognosis in KIRC. creator: Wenbiao Zhu creator: Huiming Jiang creator: Shoucheng Xie creator: Huanqin Xiao creator: Qinghua Liu creator: Nanhui Chen creator: Pei Wan creator: Shanming Lu uri: https://doi.org/10.7717/peerj.12086 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Zhu et al. title: Phylogenetic analysis of a new morphological dataset elucidates the evolutionary history of Crocodylia and resolves the long-standing gharial problem link: https://peerj.com/articles/12094 last-modified: 2021-09-06 description: First appearing in the latest Cretaceous, Crocodylia is a clade of semi-aquatic, predatory reptiles, defined by the last common ancestor of extant alligators, caimans, crocodiles, and gharials. Despite large strides in resolving crocodylian interrelationships over the last three decades, several outstanding problems persist in crocodylian systematics. Most notably, there has been persistent discordance between morphological and molecular datasets surrounding the affinities of the extant gharials, Gavialis gangeticus and Tomistoma schlegelii. Whereas molecular data consistently support a sister taxon relationship, in which they are more closely related to crocodylids than to alligatorids, morphological data indicate that Gavialis is the sister taxon to all other extant crocodylians. Here we present a new morphological dataset for Crocodylia based on a critical reappraisal of published crocodylian character data matrices and extensive firsthand observations of a global sample of crocodylians. This comprises the most taxonomically comprehensive crocodylian dataset to date (144 OTUs scored for 330 characters) and includes a new, illustrated character list with modifications to the construction and scoring of characters, and 46 novel characters. Under a maximum parsimony framework, our analyses robustly recover Gavialis as more closely related to Tomistoma than to other extant crocodylians for the first time based on morphology alone. This result is recovered regardless of the weighting strategy and treatment of quantitative characters. However, analyses using continuous characters and extended implied weighting (with high k-values) produced the most resolved, well-supported, and stratigraphically congruent topologies overall. Resolution of the gharial problem reveals that: (1) several gavialoids lack plesiomorphic features that formerly drew them towards the stem of Crocodylia; and (2) more widespread similarities occur between species traditionally divided into tomistomines and gavialoids, with these interpreted here as homology rather than homoplasy. There remains significant temporal incongruence regarding the inferred divergence timing of the extant gharials, indicating that several putative gavialids (‘thoracosaurs’) are incorrectly placed and require future re-appraisal. New alligatoroid interrelationships include: (1) support for a North American origin of Caimaninae in the latest Cretaceous; (2) the recovery of the early Paleogene South American taxon Eocaiman as a ‘basal’ alligatoroid; and (3) the paraphyly of the Cenozoic European taxon Diplocynodon. Among crocodyloids, notable results include modifications to the taxonomic content of Mekosuchinae, including biogeographic affinities of this clade with latest Cretaceous–early Paleogene Asian crocodyloids. In light of our new results, we provide a comprehensive review of the evolutionary and biogeographic history of Crocodylia, which included multiple instances of transoceanic and continental dispersal. creator: Jonathan P. Rio creator: Philip D. Mannion uri: https://doi.org/10.7717/peerj.12094 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Rio and Mannion title: Comparative analysis morphology, anatomical structure and transcriptional regulatory network of chlorophyll biosynthesis in Oryza longistaminata, O. sativa and their F1 generation link: https://peerj.com/articles/12099 last-modified: 2021-09-06 description: Oryza longistaminata, a perennial wild species, is widely distributed in the African continent. It has strong tolerance to biotic and abiotic stresses, and high biomass production on poor soils. Chlorophyll biosynthesis is important for photosynthesis in rice. However, the chlorophyll biosynthesis and related gene profiles of O. longistaminata and its descendants remained unclear. Here, the F1 generation of O. sativa and O. longistaminata were obtained. Then, the comparative analysis morphology, anatomical structure, and transcriptional regulatory networks of chlorophyll biosynthesis were detected and analyzed. Results showed that the F1 generation has obvious long awn, similar with that of the male parent. The purple color of the long awn is different from that of the male parent. Microstructural results showed that the flag leaves of F1 have large mesophyll cell gaps in the upper- and lower-positions, small mesophyll cell gaps in the middle position, and more chloroplasts. Increased chlorophyll content was also observed in the F1 generation. In the lower-position flag leaves, the total chlorophyll contents of F1 were 1.55 and 1.5 times those of O. sativa and O. longistaminata, respectively. POR, MgCH and HEMA1 showed higher expression levels than the other related genes selected in the chlorophyll biosynthesis pathway. The HEMA1 expression level in the middle-position flag leaves of O. longistaminata was the highest, and it was 2.83 and 2.51 times that of O. sativa and F1, respectively. The expression level of DVR gene in lower-position flag leaves of F1 were 93.16% and 95.06% lower than those of O. sativa and O. longistaminata, respectively. This study provided a potential reference for studying the photosynthesis and heterosis utilization of O. longistaminata. creator: Zhihang Hu creator: Xinyu Chen creator: Liexiang Huangfu creator: Shaobo Shao creator: Xiang Tao creator: Lishuang Song creator: Wenzhi Tong creator: Chuan-Deng Yi uri: https://doi.org/10.7717/peerj.12099 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Hu et al. title: Detection of spreader nodes in human-SARS-CoV protein-protein interaction network link: https://peerj.com/articles/12117 last-modified: 2021-09-06 description: The entire world is witnessing the coronavirus pandemic (COVID-19), caused by a novel coronavirus (n-CoV) generally distinguished as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 promotes fatal chronic respiratory disease followed by multiple organ failure, ultimately putting an end to human life. International Committee on Taxonomy of Viruses (ICTV) has reached a consensus that SARS-CoV-2 is highly genetically similar (up to 89%) to the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), which had an outbreak in 2003. With this hypothesis, current work focuses on identifying the spreader nodes in the SARS-CoV-human protein–protein interaction network (PPIN) to find possible lineage with the disease propagation pattern of the current pandemic. Various PPIN characteristics like edge ratio, neighborhood density, and node weight have been explored for defining a new feature spreadability index by which spreader proteins and protein–protein interaction (in the form of network edges) are identified. Top spreader nodes with a high spreadability index have been validated by Susceptible-Infected-Susceptible (SIS) disease model, first using a synthetic PPIN followed by a SARS-CoV-human PPIN. The ranked edges highlight the path of entire disease propagation from SARS-CoV to human PPIN (up to level-2 neighborhood). The developed network attribute, spreadability index, and the generated SIS model, compared with the other network centrality-based methodologies, perform better than the existing state-of-art. creator: Sovan Saha creator: Piyali Chatterjee creator: Mita Nasipuri creator: Subhadip Basu uri: https://doi.org/10.7717/peerj.12117 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Saha et al. title: VGEA: an RNA viral assembly toolkit link: https://peerj.com/articles/12129 last-modified: 2021-09-06 description: Next generation sequencing (NGS)-based studies have vastly increased our understanding of viral diversity. Viral sequence data obtained from NGS experiments are a rich source of information, these data can be used to study their epidemiology, evolution, transmission patterns, and can also inform drug and vaccine design. Viral genomes, however, represent a great challenge to bioinformatics due to their high mutation rate and forming quasispecies in the same infected host, bringing about the need to implement advanced bioinformatics tools to assemble consensus genomes well-representative of the viral population circulating in individual patients. Many tools have been developed to preprocess sequencing reads, carry-out de novo or reference-assisted assembly of viral genomes and assess the quality of the genomes obtained. Most of these tools however exist as standalone workflows and usually require huge computational resources. Here we present (Viral Genomes Easily Analyzed), a Snakemake workflow for analyzing RNA viral genomes. VGEA enables users to map sequencing reads to the human genome to remove human contaminants, split bam files into forward and reverse reads, carry out de novo assembly of forward and reverse reads to generate contigs, pre-process reads for quality and contamination, map reads to a reference tailored to the sample using corrected contigs supplemented by the user’s choice of reference sequences and evaluate/compare genome assemblies. We designed a project with the aim of creating a flexible, easy-to-use and all-in-one pipeline from existing/stand-alone bioinformatics tools for viral genome analysis that can be deployed on a personal computer. VGEA was built on the Snakemake workflow management system and utilizes existing tools for each step: fastp (Chen et al., 2018) for read trimming and read-level quality control, BWA (Li & Durbin, 2009) for mapping sequencing reads to the human reference genome, SAMtools (Li et al., 2009) for extracting unmapped reads and also for splitting bam files into fastq files, IVA (Hunt et al., 2015) for de novo assembly to generate contigs, shiver (Wymant et al., 2018) to pre-process reads for quality and contamination, then map to a reference tailored to the sample using corrected contigs supplemented with the user’s choice of existing reference sequences, SeqKit (Shen et al., 2016) for cleaning shiver assembly for QUAST, QUAST (Gurevich et al., 2013) to evaluate/assess the quality of genome assemblies and MultiQC (Ewels et al., 2016) for aggregation of the results from fastp, BWA and QUAST. Our pipeline was successfully tested and validated with SARS-CoV-2 (n = 20), HIV-1 (n = 20) and Lassa Virus (n = 20) datasets all of which have been made publicly available. VGEA is freely available on GitHub at: https://github.com/pauloluniyi/VGEA under the GNU General Public License. creator: Paul E. Oluniyi creator: Fehintola Ajogbasile creator: Judith Oguzie creator: Jessica Uwanibe creator: Adeyemi Kayode creator: Anise Happi creator: Alphonsus Ugwu creator: Testimony Olumade creator: Olusola Ogunsanya creator: Philomena Ehiaghe Eromon creator: Onikepe Folarin creator: Simon D.W. Frost creator: Jonathan Heeney creator: Christian T. Happi uri: https://doi.org/10.7717/peerj.12129 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Oluniyi et al. title: Comprehensive analysis of abnormal expression, prognostic value and oncogenic role of the hub gene FN1 in pancreatic ductal adenocarcinoma via bioinformatic analysis and in vitro experiments link: https://peerj.com/articles/12141 last-modified: 2021-09-06 description: BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most commonly diagnosed cancers with a poor prognosis worldwide. Although the treatment of PDAC has made great progress in recent years, the therapeutic effects are still unsatisfactory. Methods. In this study, we identified differentially expressed genes (DEGs) between PDAC and normal pancreatic tissues based on four Gene Expression Omnibus (GEO) datasets (GSE15471, GSE16515, GSE28735 and GSE71729). A protein–protein interaction (PPI) network was established to evaluate the relationship between the DEGs and to screen hub genes. The expression levels of the hub genes were further validated through the Gene Expression Profiling Interactive Analysis (GEPIA), ONCOMINE and Human Protein Atlas (HPA) databases, as well as the validation GEO dataset GSE62452. Additionally, the prognostic values of the hub genes were evaluated by Kaplan–Meier plotter and the validation GEO dataset GSE62452. Finally, the mechanistic roles of the most remarkable hub genes in PDAC were examined through in vitro experiments.ResultsWe identified the following nine hub genes by performing an integrated bioinformatics analysis: COL1A1, COL1A2, FN1, ITGA2, KRT19, LCN2, MMP9, MUC1 and VCAN. All of the hub genes were significantly upregulated in PDAC tissues compared with normal pancreatic tissues. Two hub genes (FN1 and ITGA2) were associated with poor overall survival (OS) rates in PDAC patients. Finally, in vitro experiments indicated that FN1 plays vital roles in PDAC cell proliferation, colony formation, apoptosis and the cell cycle.ConclusionsIn summary, we identified two hub genes that are associated with the expression and prognosis of PDAC. The oncogenic role of FN1 in PDAC was first illustrated by performing an integrated bioinformatic analysis and in vitro experiments. Our results provide a fundamental contribution for further research aimed finding novel therapeutic targets for overcoming PDAC. creator: Xiaohua Lei creator: Guodong Chen creator: Jiangtao Li creator: Wu Wen creator: Jian Gong creator: Jie Fu uri: https://doi.org/10.7717/peerj.12141 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Lei et al. title: An evaluation of factors affecting pain during transrectal ultrasonographic prostate biopsy: a real-life scenario in a retrospective cohort study link: https://peerj.com/articles/12144 last-modified: 2021-09-06 description: BackgroundPeriprostatic infiltration anesthesia (PPIA) and intrarectal topical anesthesia (IRTA) are recommended methods to control pain in transrectal ultrasonographic prostate biopsy (TRUS-Bx). This study evaluates the factors affecting pain during TRUS-Bx, considering the pathologies involved in anorectal pain etiology and comparing the effectiveness of local anesthesia techniques in providing patient comfort.Material and MethodsWe retrospectively evaluated 477 consecutive patients with TRUS-Bx for elevated Prostate Specific Antigen (PSA), abnormal rectal examination findings, or both. Patients were grouped as local anesthesia methods for pain control during TRUS-Bx. Both groups were compared in terms of age, body mass index, clinical T stage, PSA, prostate volume, number of biopsy cores, type of anesthesia, previous biopsy history, and presence of prostate cancer. We used a visual analog pain scale (VAS) to evaluate the patient’s pain status; pre-procedure (VAS-0), during probe insertion (VAS-I), administration of anesthetic (VAS-A), and simultaneous with the biopsy procedure itself (VAS-Bx). For PPIA and IRTA, 4 ml lidocaine 20 mg/ml injection and 5 g 5% prilocaine-5% lidocaine cream was used, respectively.ResultsThe PPIA was used 74.2% (n = 354) and IRTA was used for 25.8% (n = 123) patients. VAS-0, VAS-I, and VAS-A scores are similar between groups. VAS-Bx was significantly higher in the IRTA than in the PPIA (3.37 ± 0.18 vs. 2.36 ± 0.12 p > 0.001). Clinical T stage (OR: 0.59), number of biopsy cores (OR: 1.80), and type of anesthesia application (OR: 2.65) were independent variables on TRUS-Bx for pain.ConclusionThree factors play roles as independent variables associated with the pain in TRUS-Bx; abnormal rectal examination findings, collection of more than twelve core samples during the biopsy, and the type of anesthesia used. Compared with PPIA, IRTA does not improve pain related to probe insertion, and using IRTA results in higher pain scores for biopsy-related pain. Thus, we recommend a PPIA to lower biopsy-related pain. creator: Oğuz Özden Cebeci creator: Alp Ozkan uri: https://doi.org/10.7717/peerj.12144 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Cebeci and Ozkan title: Beetle bombing always deters praying mantises link: https://peerj.com/articles/11657 last-modified: 2021-09-03 description: Some animals have evolved chemical weapons to deter predators. Bombardier beetles (Coleoptera: Carabidae: Brachininae: Brachinini) can eject toxic chemicals at temperatures of 100 °C from the tips of their abdomens, ‘bombing’ the attackers. Although some bombardier beetles can reportedly deter predators, few studies have tested whether bombing is essential for successful defence. Praying mantises (Mantodea) are ambush predators that attack various arthropods. However, it is unclear whether bombardier beetles deter mantises. To test the defensive function of bombing against praying mantises, I observed three mantis species, Tenodera sinensis, Tenodera angustipennis, and Hierodula patellifera (Mantidae), attacking the bombardier beetle Pheropsophus jessoensis (Carabidae: Brachininae: Brachinini) under laboratory conditions. All mantises easily caught the beetles using their raptorial forelegs, but released them immediately after being bombed. All of the counterattacked mantises were observed to groom the body parts sprayed with hot chemicals after releasing the beetles. When treated P. jessoensis that were unable to eject hot chemicals were provided, all mantises successfully caught and devoured the treated beetles. Therefore, bombing is essential for the successful defence of P. jessoensis against praying mantises. Consequently, P. jessoensis can always deter mantises. creator: Shinji Sugiura uri: https://doi.org/10.7717/peerj.11657 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Sugiura title: Clinicopathological value of long non-coding RNA profiles in gastrointestinal stromal tumor link: https://peerj.com/articles/11946 last-modified: 2021-09-03 description: BackgroundLong non-coding RNAs (lncRNAs) have been implicated in diagnosis and prognosis in various cancers. However, few lncRNA signatures have been established for prediction of gastrointestinal stromal tumors (GIST). We aimed to explore a lncRNA signature profile that associated with clinical relevance by mining data from Gene Expression Ominus (GEO) and Surveillance, Epidemiology, and End Results (SEER) Program.MethodsUsing a lncRNA-mining approach, we performed non-negative matrix factorization (NMF) consensus algorithm in Gastrointestinal stromal tumors (GISTs) cohorts (61 patients from GSE8167 and GSE17743) to cluster LncRNA expression profiles. Comparative markers selection, and Gene Set Enrichment Analysis (GSEA) algorithm were performed between distinct molecular subtypes of GIST. The survival rate of GIST patients from SEER stratified by gender were compared by Kaplan–Meier method and log-rank analysis. lncRNA-mRNA co-expression analysis was performed by Pearson correlation coefficients (PCC) using R package LINC. Somatic copy number alterations of GIST patients (GSE40966) were analyzed via web server GenePattern GISTIC2 algorithm.ResultsA total of four lncRNA molecular subtypes of GIST were identified with distinct biological pathways and clinical characteristics. LncRNA expression profiles well clustered the GIST samples into small size (<5 mm) and large size tumors (>5 mm), which is a fundamental index for GIST malignancy diagnosis. Several lncRNAs with abundant expression (LRRC75A-AS1, HYMAI, NEAT1, XIST and FTX) were closely associated with tumor size, which may suggest to be biomarkers for the GIST malignancy. Particularly, LRRC75A-AS1 was positively associated with tumor diameters and suggested an oncogene in GIST. Co-expression analysis suggested that chromosome region 17p11.2–p12 may contribute to the oncogenic process in malignant GIST. Interestingly, the gender had a strong influence on clustering by lncRNA expression profile. Data from the Surveillance, Epidemiology, and End Results (SEER) Program were further explored and 7983 patients who were diagnosed with GISTs from 1973 to 2014 were enrolled for analysis. The results also showed the favorable prognosis for female patients. The survival rate between male and female with GIST was statistically significant (P < 0.0001). Gene set enrichment analysis (GSEA) indicated distinct pathways between female and male, and malignant GIST was associated with several cancer metabolism and cell cycle associated pathways.ConclusionsThis lncRNAs-based classification for GISTs may provide a molecular classification applicable to individual GIST that has implications to influence lncRNA markers selection and prediction of tumor progression. creator: Yan Zhao creator: Xinxin Liu creator: Keshuai Xiao creator: Liwen Wang creator: Yuping Li creator: Mingyun Kan creator: Zhiwei Jiang uri: https://doi.org/10.7717/peerj.11946 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Zhao et al. title: Expression and prognosis of CDC45 in cervical cancer based on the GEO database link: https://peerj.com/articles/12114 last-modified: 2021-09-03 description: Cervical cancer is one of the most common malignant tumors in women, and its morbidity and mortality are increasing year by year worldwide. Therefore, an urgent and challenging task is to identify potential biomarkers for cervical cancer. This study aims to identify the hub genes based on the GEO database and then validate their prognostic values in cervical cancer by multiple databases. By analysis, we obtained 83 co-expressed differential genes from the GEO database (GSE63514, GSE67522 and GSE39001). GO and KEGG enrichment analysis showed that these 83 co-expressed it mainly involved differential genes in DNA replication, cell division, cell cycle, etc.. The PPI network was constructed and top 10 genes with protein-protein interaction were selected. Then, we validated ten genes using some databases such as TCGA, GTEx and oncomine. Survival analysis demonstrated significant differences in CDC45, RFC4, TOP2A. Differential expression analysis showed that these genes were highly expressed in cervical cancer tissues. Furthermore, univariate and multivariate cox regression analysis indicated that CDC45 and clinical stage IV were independent prognostic factors for cervical cancer. In addition, the HPA database validated the protein expression level of CDC45 in cervical cancer. Further studies investigated the relationship between CDC45 and tumor-infiltrating immune cells via CIBERSORT. Finally, gene set enrichment analysis (GSEA) showed CDC45 related genes were mainly enriched in cell cycle, chromosome, catalytic activity acting on DNA, etc. These results suggested CDC45 may be a potential biomarker associated with the prognosis of cervical cancer. creator: Zikang He creator: Xiaojin Wang creator: Zhiming Yang creator: Ying Jiang creator: Luhui Li creator: Xingyun Wang creator: Zheyao Song creator: Xiuli Wang creator: Jiahui Wan creator: Shijun Jiang creator: Naiwen Zhang creator: Rongjun Cui uri: https://doi.org/10.7717/peerj.12114 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 He et al. title: Analysis of microRNA expression in CD133 positive cancer stem‑like cells of human osteosarcoma cell line MG-63 link: https://peerj.com/articles/12115 last-modified: 2021-09-03 description: Osteosarcoma (OS) is a primary malignant tumor of bone occurring in young adults. OS stem cells (OSCs) play an important role in the occurrence, growth, metastasis, drug resistance and recurrence of OS. CD133 is an integral membrane glycoprotein, which has been identified as an OSC marker. However, the mechanisms of metastasis, chemoresistance, and progression in CD133(+) OSCs need to be further explored. In this study, we aim to explore differences in miRNA levels between CD133(+) and CD133(−) cells from the MG-63 cell line. We found 20 differentially expressed miRNAs (DEmiRNAs) (16 upregulated and 4 downregulated) in CD133(+) cells compared with CD133(−) cells. Hsa-miR-4485-3p, hsa-miR-4284 and hsa-miR-3656 were the top three upregulated DEmiRNAs, while hsa-miR-487b-3p, hsa-miR-493-5p and hsa-miR-431-5p were the top three downregulated DEmiRNAs. In addition, RT-PCR analysis confirmed that the expression levels of hsa-miR-4284, hsa-miR-4485-3p and hsa-miR-3656 were significantly increased, while the expression levels of hsa-miR-487b-3p, hsa-miR-493-5p, and hsa-miR-431-5p were significantly decreased in CD133(+) cells compared with CD133(−) cells. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that predicted or validated target genes for all 20 DEmiRNAs or the selected 6 DEmiRNAs participated in the “PI3K-Akt signaling pathway,” “Wnt signaling pathway,” “Rap1 signaling pathway,” “Cell cycle” and “MAPK signaling pathway”. Among the selected six DEmiRNAs, miR-4284 was especially interesting. MiR-4284 knockdown significantly reduced the sphere forming capacity of CD133(+) OS cells. The number of invasive CD133(+) OS cells was markedly decreased after miR-4284 knockdown. In addition, miR-4284 knockdown increased the p-β-catenin levels in CD133(+) OS cells. In conclusion, RNA-seq analysis revealed DEmiRNAs between CD133(+) and CD133(−) cells. MiRNAs might play significant roles in the function of OSCs and could serve as targets for OS treatment. MiR-4284 prompted the self-renewal and invasion of OSCs. The function of miR-4284 might be associated with the Wnt signaling pathway. creator: Xiong Shu creator: Weifeng Liu creator: Huiqi Liu creator: Hui Qi creator: Chengai Wu creator: Yu-Liang Ran uri: https://doi.org/10.7717/peerj.12115 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Shu et al. title: The LEAF questionnaire is a good screening tool for the identification of the Female Athlete Triad/Relative Energy Deficiency in Sport among young football players link: https://peerj.com/articles/12118 last-modified: 2021-09-03 description: BackgroundIt has been noticed that Female Athlete Triad (Fat) and Relative Energy Deficiency (Red-S) in Sport are characterized by the symptoms of impaired endocrine-metabolic function and bone health in female athletes. In addition, it may be evaluated with a qualitative tool, such as Low Energy Availability in Females questionnaire (LEAF-Q) and quantitative measurements: bone mineral density (BMD), resting energy expenditure (REE), body composition, 24-hour dietary recall.MethodsThe aim of this study was to assess the prevalence of Triad and Red-S using the LEAF-Q in youth female football players. Additionally, the difference in the BMD, body composition, REE and energy intake (EI) were assessed between the Triad/Red-S risk and not at-risk groups.ResultsAlmost two thirds (64.7%) of participants are classified as being at-risk for the triad according to their LEAF-Q scores. There were no statistically significant differences (p > 0.05) between most of the values among children from the analyzed groups. There was a statistically significant difference (p < 0.001) between the EI values among girls from the two analyzed groups: at-risk (1,773.18 kcal ±  232.57) and not at-risk (2,054.00 kcal ±  191.39). Girls who did not meet the energy intake recommendations were 10.00 as likely to be in the Triad/Red-S risk group.ConclusionEarly identification of Fat/Red-S symptoms by screening tools such as the LEAF questionnaire is important in protecting young athletes from long-term damage due to the progression of the risk factors associated with the Fat/Red-S. creator: Edyta Łuszczki creator: Pawel Jagielski creator: Anna Bartosiewicz creator: Maciej Kuchciak creator: Katarzyna Dereń creator: Artur Stolarczyk creator: Paweł Pakosz creator: Lukasz Oleksy uri: https://doi.org/10.7717/peerj.12118 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Łuszczki et al. title: Spatial distribution of parrotfishes and groupers in an Okinawan coral reef: size-related associations in relation to habitat characteristics link: https://peerj.com/articles/12134 last-modified: 2021-09-03 description: Parrotfishes (Labridae: Scarini) and groupers (Epinephelidae) are important fish groups that are regarded as the fisheries targets of primary importance in coral reefs. In order to establish ecosystem-based management of these two fish groups, clarifying the spatial distribution relative to habitat characteristics is of central importance. The present study investigated the spatial distributions of 12 parrotfishes species and seven groupers species in relation to environmental characteristics in an Okinawan coral reef. Ten out of the 12 parrotfish species and all seven grouper species showed species-specific spatial distributions. Four substrate types in the inner reefs (branching Acropora, bottlebrush Acropora, dead branching Acropora, and dead bottlebrush Acropora), three substrate types in the exposed reefs (massive coral, other coral, and calcium carbonate substratum), and water depth showed significant associations with the spatial distribution of fishes. Among the 12 parrotfish species, two species (Scarus spinus and S. forsteni) and four species (S. psittacus, S. hypselopterus, S. dimidiatus and S. ghobban) were primarily found in exposed reefs and inner reefs, respectively. Among the seven grouper species, two species (Cephalopholis argus and C. urodeta) and two other species (C. miniata and Epinephelus ongus) were primarily found in exposed reefs and inner reefs, respectively. Size-related spatial distribution was also found for three parrotfish species (Chlorurus microrhinos, Scarus rivulatus and S. hypselopterus), indicating that smaller-sized and larger-sized individuals were respectively found at sites with greater coverage of substrates with fine structure (live bottlebrush Acropora and dead bottlebrush Acropora) and coarse structure (live branching Acropora, dead branching Acropora and calcium carbonate substratum). The present study suggested that the spatial distribution of parrotfishes and groupers is not necessarily associated with the higher coverage of living corals, but positively associated with high substrate complexity. Thus, actual spatial distributional patterns of species should be considered to select candidate sites for protection and conservation for the two fish groups. creator: Atsushi Nanami uri: https://doi.org/10.7717/peerj.12134 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Nanami title: Organic soil amendments using vermicomposts under inoculation of N2-fixing bacteria for sustainable rice production link: https://peerj.com/articles/10833 last-modified: 2021-09-02 description: Organic and biological fertilizers are considered as a very important source of plant nutrients. A field experiment was conducted during 2017−2018 in paddy soil to investigate the effect of vermicomposting of cattle manure mixture with Azolla and rice straw on soil microbial activity, nutrient uptake, and grain yield under inoculation of N2-fixing bacteria. Experimental factors consisted of organic amendments at six levels (vermicomposts prepared from manure (VM); manure + rice straw (VRM); manure + Azolla mixture (VAM); manure + rice straw + Azolla mixture (VRAM); raw manure without vermicomposting (M), and a control) and N2-fixing bacteria at three levels (Azotobacter chroococcum, Azospirillum brasilence, and non−inoculation). The results showed that, vermicompost treatments compared to control and raw manure significantly increased the number and biomass−C of soil microorganisms, urease activity, number of tillers hill−1, phosphorus (P) and potassium (K) uptake, and grain and protein yield. Inoculation of plants with N2-fixing bacteria, especially Azotobacter increased the efficiency of organic amendments, so that the maximum urease activity, soil microbial activity, P and N uptake, and grain yield (4,667 (2017) and 5,081 (2018) kg/h) were observed in vermicompost treatments containing Azolla (VAM and VRAM) under inoculation with Azotobacter. The results of the study suggested that, using an organic source along with inoculation with appropriate N2-fixing bacteria for vermicompost has a great effect on enzyme activity, soil biology, nutrient uptake and grain yield has a synergistic interaction on agronomic traits under flooded conditions. Therefore, this nutrient method can be used as one of the nutrient management strategies in the sustainable rice production. creator: Mehdi Ghadimi creator: Alireza Sirousmehr creator: Mohammad Hossein Ansari creator: Ahmad Ghanbari uri: https://doi.org/10.7717/peerj.10833 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Ghadimi et al. title: Weak spatial-genetic structure in a native invasive, the southern pine beetle (Dendroctonus frontalis), across the eastern United States link: https://peerj.com/articles/11947 last-modified: 2021-09-02 description: The southern pine beetle, Dendroctonus frontalis, is a native pest of pine trees that has recently expanded its range into the northeastern United States. Understanding its colonization, dispersal, and connectivity will be critical for mitigating negative economic and ecological impacts in the newly invaded areas. Characterization of spatial-genetic structure can contribute to this; however, previous studies have reached different conclusions about regional population genetic structure, with one study reporting a weak east-west pattern, and the most recent reporting an absence of structure. Here we systematically assessed several explanations for the absence of spatial-genetic structure. To do this, we developed nine new microsatellite markers and combined them with an existing 24-locus data matrix for the same individuals. We then reanalyzed this full dataset alongside datasets in which certain loci were omitted with the goal of creating more favorable signal to noise ratios. We also partitioned the data based on the sex of D. frontalis individuals, and then employed a broad suite of genotypic clustering and isolation-by-distance (IBD) analyses. We found that neither inadequate information content in the molecular marker set, nor unfavorable signal-to-noise ratio, nor insensitivity of the analytical approaches could explain the absence of structure. Regardless of dataset composition, there was little evidence for clusters (i.e., distinct geo-genetic groups) or clines (i.e., gradients of increasing allele frequency differences over larger geographic distances), with one exception: significant IBD was repeatedly detected using an individual-based measure of relatedness whenever datasets included males (but not for female-only datasets). This is strongly indicative of broad-scale female-biased dispersal, which has not previously been reported for D. frontalis, in part owing to logistical limitations of direct approaches (e.g., capture-mark-recapture). Weak spatial-genetic structure suggests long-distance connectivity and that gene flow is high, but additional research is needed to understand range expansion and outbreak dynamics in this species using alternate approaches. creator: Ryan C. Garrick creator: Ísis C. Arantes creator: Megan B. Stubbs creator: Nathan P. Havill uri: https://doi.org/10.7717/peerj.11947 license: https://creativecommons.org/publicdomain/zero/1.0/ rights: ©2021 Garrick et al. title: Genomic analysis of early transmissibility assessment of the D614G mutant strain of SARS-CoV-2 in travelers returning to Taiwan from the United States of America link: https://peerj.com/articles/11991 last-modified: 2021-09-02 description: BackgroundThere is a global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Information on viral genomics is crucial for understanding global dispersion and for providing insight into viral pathogenicity and transmission. Here, we characterized the SARS-CoV-2 genomes isolated from five travelers who returned to Taiwan from the United States of America (USA) between March and April 2020.MethodsHaplotype network analysis was performed using genome-wide single-nucleotide variations to trace potential infection routes. To determine the genetic variations and evolutionary trajectory of the isolates, the genomes of isolates were compared to those of global virus strains from GISAID. Pharyngeal specimens were confirmed to be SARS-CoV-2-positive by RT-PCR. Direct whole-genome sequencing was performed, and viral assemblies were subsequently uploaded to GISAID. Comparative genome sequence and single-nucleotide variation analyses were performed.ResultsThe D614G mutation was identified in imported cases, which separated into two clusters related to viruses originally detected in the USA. Our findings highlight the risk of spreading SARS-CoV-2 variants through air travel and the need for continued genomic tracing for the epidemiological investigation and surveillance of SARS-CoV-2 using viral genomic data.ConclusionsContinuous genomic surveillance is warranted to trace virus circulation and evolution in different global settings during future outbreaks. creator: Ming-Jr Jian creator: Hsing-Yi Chung creator: Chih-Kai Chang creator: Shan-Shan Hsieh creator: Jung-Chung Lin creator: Kuo-Ming Yeh creator: Chien-Wen Chen creator: Feng-Yee Chang creator: Kuo-Sheng Hung creator: Ming-Tsan Liu creator: Ji-Rong Yang creator: Tein-Yao Chang creator: Sheng-Hui Tang creator: Cherng-Lih Perng creator: Hung-Sheng Shang uri: https://doi.org/10.7717/peerj.11991 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Jian et al. title: The spleen bacteriome of wild rodents and shrews from Marigat, Baringo County, Kenya link: https://peerj.com/articles/12067 last-modified: 2021-09-02 description: BackgroundThere is a global increase in reports of emerging diseases, some of which have emerged as spillover events from wild animals. The spleen is a major phagocytic organ and can therefore be probed for systemic microbiome. This study assessed bacterial diversity in the spleen of wild caught small mammals so as to evaluate their utility as surveillance tools for monitoring bacteria in an ecosystem shared with humans.MethodsFifty-four small mammals (rodents and shrews) were trapped from different sites in Marigat, Baringo County, Kenya. To characterize their bacteriome, DNA was extracted from their spleens and the V3–V4 regions of the 16S rRNA amplified and then sequenced on Illumina MiSeq. A non-target control sample was used to track laboratory contaminants. Sequence data was analyzed with Mothur v1.35, and taxomy determined using the SILVA database. The Shannon diversity index was used to estimate bacterial diversity in each animal and then aggregated to genus level before computing the means. Animal species within the rodents and shrews were identified by amplification of mitochondrial cytochrome b (cytb) gene followed by Sanger sequencing. CLC workbench was used to assemble the cytb gene sequences, after which their phylogenetic placements were determined by querying them against the GenBank nucleotide database.Resultscytb gene sequences were generated for 49/54 mammalian samples: 38 rodents (Rodentia) and 11 shrews (Eulipotyphyla). Within the order Rodentia, 21 Acomys, eight Mastomys, six Arvicanthis and three Rattus were identified. In the order Eulipotyphyla, 11 Crucidura were identified. Bacteria characterization revealed 17 phyla that grouped into 182 genera. Of the phyla, Proteobacteria was the most abundant (67.9%). Other phyla included Actinobacteria (16.5%), Firmicutes (5.5%), Chlamydiae (3.8%), Chloroflexi (2.6%) and Bacteroidetes (1.3%) among others. Of the potentially pathogenic bacteria, Bartonella was the most abundant (45.6%), followed by Anaplasma (8.0%), Methylobacterium (3.5%), Delftia (3.8%), Coxiella (2.6%), Bradyrhizobium (1.6%) and Acinetobacter (1.1%). Other less abundant (<1%) and potentially pathogenic included Ehrlichia, Rickettsia, Leptospira, Borrelia, Brucella, Chlamydia and Streptococcus. By Shannon diversity index, Acomys spleens carried more diverse bacteria (mean Shannon diversity index of 2.86, p = 0.008) compared to 1.77 for Crocidura, 1.44 for Rattus, 1.40 for Arvicathis and 0.60 for Mastomys.ConclusionThis study examined systemic bacteria that are filtered by the spleen and the findings underscore the utility of 16S rRNA deep sequencing in characterizing complex microbiota that are potentially relevant to one health issues. An inherent problem with the V3-V4 region of 16S rRNA is the inability to classify bacteria reliably beyond the genera. Future studies should utilize the newer long read methods of 16S rRNA analysis that can delimit the species composition. creator: Rehema Liyai creator: Gathii Kimita creator: Clement Masakhwe creator: David Abuom creator: Beth Mutai creator: David Miruka Onyango creator: John Waitumbi uri: https://doi.org/10.7717/peerj.12067 license: https://creativecommons.org/publicdomain/zero/1.0/ rights: ©2021 Liyai et al. title: Hemin attenuates response of primary rat adipocytes to adrenergic stimulation link: https://peerj.com/articles/12092 last-modified: 2021-09-02 description: Hemin is an activator of heme oxygenase-1 (HO-1), an enzyme catalyzing heme degradation. Up-regulation of HO-1 is observed in response to various pathological conditions. Moreover, pharmacological activation of HO-1 is associated with numerous beneficial effects in the organism. Hemin was shown to exert, among other, anti-diabetic and anti-obesity properties. These effects are strongly linked with adipose tissue. However, the direct influence of hemin on metabolism of the fat cells have not been explored. The present study aimed to determine the short-term effects of hemin on metabolism of the primary rat adipocytes. We focused on processes directly related to lipid accumulation, such as lipogenesis and lipolysis. For this purpose, the isolated cells were subjected for 2 h to 40 µM hemin, and effects of this compound on insulin-stimulated glucose conversion to lipids, lactate release, lipolysis induced by various stimuli, and also on the antilipolytic action of insulin were determined. It was shown that hemin did not affect insulin-induced lipogenesis and lactate release. However, hemin significantly decreased lipolysis stimulated by epinephrine. The inhibitory effect of hemin on epinephrine-induced lipolysis was not abolished in the presence of SnMP, an inhibitor of HO-1, which suggests hemin action irrespective of this enzyme. Similar inhibitory effects on epinephrine-induced lipolysis were observed in the presence of 3 and 12 mM glucose. Moreover, hemin was shown to reduce epinephrine-induced lipolysis also when glucose was replaced by alanine or by succinate. Apart from changes in epinephrine action, it was found that the lipolytic response of the adipocytes to isoproterenol was also diminished by hemin. However, hemin failed to affect lipolysis stimulated by dibutyryl-cAMP (a direct activator of protein kinase A), forskolin (an activator of adenylate cyclase), and also by DPCPX (an adenosine A1 receptor antagonist). Additionally, epinephrine-induced lipolysis was shown to be decreased by insulin, and this effect was deepened in the presence of hemin. These results indicate that short-term exposure of the adipocytes to hemin does not affect processes related to glucose metabolism, such as lipogenesis and lactate release. However, hemin was found to decrease the lipolytic response to adrenergic stimulation, which is associated with reduced lipid release from adipocytes. Moreover, our results indicate that hemin is also capable of diminishing the exaggerated lipolysis, which occurs in the presence of supraphysiological concentrations of glucose. creator: Tomasz Szkudelski creator: Karina Frąckowiak creator: Katarzyna Szkudelska uri: https://doi.org/10.7717/peerj.12092 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Szkudelski et al. title: Species diversity and community structure of crustacean zooplankton in the highland small waterbodies in Northwest Yunnan, China link: https://peerj.com/articles/12103 last-modified: 2021-09-02 description: Small waterbodies are a unique aquatic ecosystem with an increasing recognition for their important role in maintaining regional biodiversity and delivering ecosystem services. However, small waterbodies in Northwest Yunnan, one of the most concerned global biodiversity hot-spots, remain largely unknown. Here, we investigated the community structure of crustacean zooplankton and their relationships with limnological, morphometric and spatial variables in the highland small waterbodies in Northwest Yunnan in both the dry (October 2015) and rainy (June 2016) seasons. A total of 38 species of crustacean zooplankton were identified in our study, which is significantly higher than many other reported waterbodies in the Yunnan–Guizhou plateau as well as in the Yangtze River basin. This suggests that the highland small waterbodies are critical in maintaining regional zooplankton diversity in Northwest Yunnan. Meanwhile, we found limnological variables could explain most variation of crustacean zooplankton community, comparing to the morphometric and spatial variables in both the rainy and dry seasons. Our study revealed the diversity and community structure of crustacean zooplankton in the highland small waterbodies in Northwest Yunnan and highlighted the importance of small waterbodies in maintaining regional biodiversity. creator: Xing Chen creator: Qinghua Cai creator: Lu Tan creator: Shuoran Liu creator: Wen Xiao creator: Lin Ye uri: https://doi.org/10.7717/peerj.12103 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Chen et al. title: Extending density surface models to include multiple and double-observer survey data link: https://peerj.com/articles/12113 last-modified: 2021-09-02 description: Spatial models of density and abundance are widely used in both ecological research (e.g., to study habitat use) and wildlife management (e.g., for population monitoring and environmental impact assessment). Increasingly, modellers are tasked with integrating data from multiple sources, collected via different observation processes. Distance sampling is an efficient and widely used survey and analysis technique. Within this framework, observation processes are modelled via detection functions. We seek to take multiple data sources and fit them in a single spatial model. Density surface models (DSMs) are a two-stage approach: first accounting for detectability via distance sampling methods, then modelling distribution via a generalized additive model. However, current software and theory does not address the issue of multiple data sources. We extend the DSM approach to accommodate data from multiple surveys, collected via conventional distance sampling, double-observer distance sampling (used to account for incomplete detection at zero distance) and strip transects. Variance propagation ensures that uncertainty is correctly accounted for in final estimates of abundance. Methods described here are implemented in the dsm R package. We briefly analyse two datasets to illustrate these new developments. Our new methodology enables data from multiple distance sampling surveys of different types to be treated in a single spatial model, enabling more robust abundance estimation, potentially over wider geographical or temporal domains. creator: David L. Miller creator: David Fifield creator: Ewan Wakefield creator: Douglas B. Sigourney uri: https://doi.org/10.7717/peerj.12113 license: https://creativecommons.org/publicdomain/zero/1.0/ rights: title: Level of anxiety among healthcare providers during COVID-19 pandemic in Saudi Arabia: cross-sectional study link: https://peerj.com/articles/12119 last-modified: 2021-09-02 description: BackgroundThe burden of the spread of the COVID-19 pandemic has impacted widely on the healthcare providers physically and mentally. Many healthcare providers are exposed to psychological stressors due to their high risk of contracting the virus.AimsThis study aimed to measure the level of anxiety among healthcare providers during the COVID-19 pandemic in Saudi Arabia. In addition, this study aimed to measure the level of anxiety based on demographic characteristics.MethodA cross-sectional survey was employed to recruit a convenience sample of healthcare providers. A pencil and paper self-administered questionnaires were used to collect data from demographic and generalized anxiety disorder GAD-7 data. However, this study received written informed consent from participants of the study. In addition, the study was approved by the Institutional Review Board at Dr. Sulaiman Al Habib Medical Group (IRB Log No. RC20.06.88-03).ResultsA total of 650 participants were recruited, results of GAD-7 showed that 43.5%, 28.9% and 27.5% of healthcare providers in Saudi Arabia experienced mild, moderate and severe anxiety, respectively, during the COVID-19 pandemic. Results indicated that age, health specialty, nationality, and sleeping disorders before COVID-19 were associated with anxiety levels.ConclusionThe generalized anxiety among healthcare providers in Saudi Arabia was mild. Older healthcare providers were found to have a higher level of anxiety compared to other participating healthcare providers. Several factors may contribute to a higher level of anxiety including age, socioeconomic status, marital status, having chronic conditions, and sleeping disorder before the COVID-19 pandemic. To further understand the level of anxiety among healthcare providers during the COVID-19 pandemic in Saudi Arabia, longitudinal and mixed-method research is needed. creator: Abbas Al Mutair creator: Alya Al Mutairi creator: Yasmine Alabbasi creator: Abbas Shamsan creator: Sana Al-Mahmoud creator: Saad Alhumaid creator: Muhammad zeshan Arshad creator: Mansour Awad creator: Ali Rabaan uri: https://doi.org/10.7717/peerj.12119 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Al Mutair et al. title: Revisiting food-sourced vitamins for consumer diet and health needs: a perspective review, from vitamin classification, metabolic functions, absorption, utilization, to balancing nutritional requirements link: https://peerj.com/articles/11940 last-modified: 2021-09-01 description: The significant attention gained by food-sourced vitamins has provided insights into numerous current researches; for instance, the potential reversal of epigenetic age using a diet and lifestyle intervention, the balance between food and dietary supplements in the general population, the role of diet and food intake in age-related macular degeneration, and the association of dietary supplement use, nutrient intake and mortality among adults. As relevant literature about food-sourced vitamin increases, continuous synthesis is warranted. To supplement existing information, this perspective review discussed food-sourced vitamins for consumer diet and health needs, scoping from vitamin absorption, metabolic functions, utilization, to balancing nutritional requirements. Relevant literatures were identified through a search of databases like Google Scholar, Web of Science, the Interscience Online Library, ScienceDirect, and PubMed. We demonstrated that vitamins whether from plant- and animal-based sources are prerequisites for the metabolic functions of the human body. The fat- and water-soluble classification of vitamins remains consistent with their respective absorption and dissolution potentials, underpinned by numerous physiological functions. Vitamins, largely absorbed in the small intestine, have their bioavailability dependent on the food composition, its associated interactions, as well as alignment with their metabolic functions, which involves antioxidants, coenzymes, electron acceptor/donor, and hormones. Moreover, vitamin deficiencies, in every form, pose a serious threat to human health. Vitamin toxicities remain rare, but can still occur mainly from supplementation, although it appears much less in water-soluble vitamins of which some excesses get readily removed by the human body, different from the fat-soluble ones that are stored in tissues and organs. Besides discussions of absorption, transport, and cellular uptake of vitamins, this perspective review also included approaches to meeting vitamin requirements and therapeutic strategies against micronutrient deficiency and COVID-19. We have also attempted on how to strike the balance between food-sourced vitamins and dietary supplements. creator: Chigozie E. Ofoedu creator: Jude O. Iwouno creator: Ebelechukwu O. Ofoedu creator: Chika C. Ogueke creator: Victory S. Igwe creator: Ijeoma M. Agunwah creator: Arinze F. Ofoedum creator: James S. Chacha creator: Onyinye P. Muobike creator: Adedoyin O. Agunbiade creator: Njideka E. Njoku creator: Angela A. Nwakaudu creator: Nkiru E. Odimegwu creator: Onyekachi E. Ndukauba creator: Chukwuka U. Ogbonna creator: Joncer Naibaho creator: Maciej Korus creator: Charles Odilichukwu R. Okpala uri: https://doi.org/10.7717/peerj.11940 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Ofoedu et al. title: Antimicrobial resistance among Enterobacteriaceae, Staphylococcus aureus, and Pseudomonas spp. isolates from clinical specimens from a hospital in Nairobi, Kenya link: https://peerj.com/articles/11958 last-modified: 2021-09-01 description: BackgroundAntimicrobial resistance among pathogens of public health importance is an emerging problem in sub-Saharan Africa. Unfortunately, published information on the burden and patterns of antimicrobial resistance (AMR) in this region is sparse. There is evidence that the burden and patterns of AMR vary by geography and facility. Knowledge of local epidemiology of AMR is thus important for guiding clinical decisions and mitigation strategies. Therefore, the objective of this study was to determine the burden and predictors of AMR and multidrug resistance (MDR) among bacterial pathogens isolated from specimens submitted to the diagnostic laboratory of a hospital in Nairobi, Kenya.MethodsThis retrospective study used laboratory records of 1,217 clinical specimens submitted for bacterial culture and sensitivity testing at the diagnostic laboratory of The Karen Hospital in Nairobi, Kenya between 2012 and 2016. Records from specimens positive for Enterobacteriaceae, Staphylococcus aureus, or Pseudomonas spp. isolates were included for analysis. Firth logistic models, which minimize small sample bias, were used to investigate determinants of AMR and MDR of the isolates.ResultsA total of 222 specimens had bacterial growth. Most Enterobacteriaceae isolates were resistant to commonly used drugs such as penicillin/β-lactamase inhibitor combinations (91.2%) and folate pathway inhibitors (83.7%). Resistance to extended-spectrum cephalosporins was also high (52.9%). Levels of AMR and MDR for Enterobacteriaceae were 88.5% and 51%, respectively. Among S. aureus isolates, 57.1% were AMR, while 16.7% were MDR. As many as 42.1% of the Pseudomonas spp. isolates were aminoglycoside-resistant and 15% were fluoroquinolone-resistant, but none exhibited resistance to antipseudomonal carbapenems. Half of Pseudomonas spp. isolates were AMR but none were MDR. Significant predictors of MDR among Enterobacteriaceae were organism species (p = 0.002) and patient gender (p = 0.024).ConclusionsThe high levels of extended-spectrum cephalosporin resistance and MDR among Enterobacteriaceae isolates are concerning. However, the relatively low levels of MDR S. aureus, and an absence of carbapenem resistance among Pseudomonas isolates, suggests that last-line drugs are still effective against S. aureus and Pseudomonas infections. These findings are relevant for guiding evidence-based treatment decisions as well as surveillance efforts and directions for future research, and contribute to the sparse literature on AMR in sub-Saharan Africa. creator: Jennifer Lord creator: Anthony Gikonyo creator: Amos Miwa creator: Agricola Odoi uri: https://doi.org/10.7717/peerj.11958 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Lord et al. title: To culture or not to culture: a snapshot of culture-dependent and culture-independent bacterial diversity from peanut rhizosphere link: https://peerj.com/articles/12035 last-modified: 2021-09-01 description: BackgroundSequencing driven metagenomics studies have been instrumental in various aspects of microbiology including identification of newer taxa. While this culture-independent approach has its own merits and demerits, several studies have focussed on comparing it with traditional culture-dependent (CD) approach. However, most of these comparative studies rely on Sanger sequencing of complete 16S rRNA gene from pure culture colonies to determine the culturable bacterial diversity. This approach undercounts culturable diversity as only fewer isolates are selected, sequenced, and identified.MethodsIn this study, we have used an Illumina based partial 16S sequencing to identify all the microbes growing on the media and directly comparing with its culture-independent (CI) counterpart. Eight different media were used to target different organisms from soil. Diversity on these media were compared with their CI counterpart. The NGS data was analysed using DADA2 to provide more resolution to the data.ResultsIn line with studies of similar nature, current study presented higher bacterial diversity in CI approach. However, the current study reflected that a greater number of sequence variants were missed out in CI approach as compared to number of sequence variants shared with CD approach. We observed around 322 (5.98%) ASVs (Amplicon Sequence Variants) exclusively present in CD samples while, 234 (4.35%) ASVs were shared between both approaches. Most of these 322 CD exclusive ASVs were classified as Enterobacteriaceae family and Bacillus genus, with several ASVs annotated at the species level as well, and these organisms are more commonly observed in soil and were also detected in CI approach. Furthermore, 22 genera were exclusively detected in CD samples, most of which were reported from soil and water. creator: Ankit Hinsu creator: Ashvin Dumadiya creator: Anjali Joshi creator: Rohitkumar Kotadiya creator: Kavan Andharia creator: Prakash Koringa creator: Ramesh Kothari uri: https://doi.org/10.7717/peerj.12035 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Hinsu et al. title: Oral health-related quality-of-life scores differ by socioeconomic status, mother’s level of education, dental visits and severity of malocclusion in mixed dentition of eight-to-ten-year-old schoolchildren link: https://peerj.com/articles/12062 last-modified: 2021-09-01 description: ObjectiveTo determinate the association among socioeconomic status subject’s mother’s level of educational attainment, dental visits, and malocclusion in mixed dentition with on the OHRQoL of eight-to-ten-year-old children.MethodsA cross-sectional study conducted, in 2019, on Mexican children from households of different socioeconomic status (SES). The prevalence of malocclusion was evaluated using the Dental Aesthetic Index (DAI), while the SES of the participants’ households was evaluated using the three categories (corresponding to a high, middle, or low-income household) stipulated by the Consejo Nacional de Población (CONAPO or National Population Council). Oral Health-related Quality of Life (OHRQoL) was evaluated using the Child Perceptions Questionnaire (CPQ8-10). Poisson regression models were performed for the analysis of the data obtained.ResultsA total of 79.4% of the subjects presented some type of malocclusion in mixed dentition, which was, by severity, as follows: definite (31.3%); severe (25.6%); and, very severe (22.5%). The Poisson regression model revealed a greater negative impact on the following four CPQ8-10domains for children with severe/very severe malocclusion [RR]: oral symptoms [2.78]; functional limitations [2.72]; emotional well-being [2.59]; and, social well-being [3.99]. A greater impact on the four CPQ8-10domains was found for children from a low-income household than for children from a high-income (p < 0.001) household. Furthermore, poor oral hygiene, lack of dental visits, and the mother’s level of educational attainment (<9 years) were found to have a negative impact on OHRQoL.ConclusionThe findings of the present study demonstrated that the severity of malocclusion was associated with a greater negative impact on the OHRQoL of children, while those children who face greater health inequalities are likely to report a greater negative impact on their OHRQoL. creator: Alvaro García Pérez creator: Álvaro Edgar González-Aragón Pineda creator: Hilda Gonzalez Olivares uri: https://doi.org/10.7717/peerj.12062 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 García Pérez et al. title: The biomechanical characteristics of the strongman atlas stone lift link: https://peerj.com/articles/12066 last-modified: 2021-09-01 description: BackgroundThe atlas stone lift is a popular strongman exercise where athletes are required to pick up a large, spherical, concrete stone and pass it over a bar or place it on to a ledge. The aim of this study was to use ecologically realistic training loads and set formats to (1) establish the preliminary biomechanical characteristics of athletes performing the atlas stone lift; (2) identify any biomechanical differences between male and female athletes performing the atlas stone lift; and (3) determine temporal and kinematic differences between repetitions of a set of atlas stones of incremental mass.MethodsKinematic measures of hip, knee and ankle joint angle, and temporal measures of phase and repetition duration were collected whilst 20 experienced strongman athletes (female: n = 8, male: n = 12) performed three sets of four stone lifts of incremental mass (up to 85% one repetition maximum) over a fixed-height bar.ResultsThe atlas stone lift was categorised in to five phases: the recovery, initial grip, first pull, lap and second pull phase. The atlas stone lift could be biomechanically characterised by maximal hip and moderate knee flexion and ankle dorsiflexion at the beginning of the first pull; moderate hip and knee flexion and moderate ankle plantarflexion at the beginning of the lap phase; moderate hip and maximal knee flexion and ankle dorsiflexion at the beginning of the second pull phase; and maximal hip, knee extension and ankle plantarflexion at lift completion. When compared with male athletes, female athletes most notably exhibited: greater hip flexion at the beginning of the first pull, lap and second pull phase and at lift completion; and a shorter second pull phase duration. Independent of sex, first pull and lap phase hip and ankle range of motion (ROM) were generally smaller in repetition one than the final three repetitions, while phase and total repetition duration increased throughout the set. Two-way interactions between sex and repetition were identified. Male athletes displayed smaller hip ROM during the second pull phase of the first three repetitions when compared with the final repetition and smaller hip extension at lift completion during the first two repetitions when compared with the final two repetitions. Female athletes did not display these between-repetition differences.ConclusionsSome of the between-sex biomechanical differences observed were suggested to be the result of between-sex anthropometric differences. Between-repetition differences observed may be attributed to the increase in stone mass and acute fatigue. The biomechanical characteristics of the atlas stone lift shared similarities with the previously researched Romanian deadlift and front squat. Strongman athletes, coaches and strength and conditioning coaches are recommended to take advantage of these similarities to achieve greater training adaptations and thus performance in the atlas stone lift and its similar movements. creator: Benjamin Hindle creator: Anna Lorimer creator: Paul Winwood creator: Daniel Brimm creator: Justin W.L. Keogh uri: https://doi.org/10.7717/peerj.12066 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Hindle et al. title: Predictors of periodontal and caries related perinatal oral healthcare, investigation of dentists’ practices: a cross-sectional study link: https://peerj.com/articles/12080 last-modified: 2021-09-01 description: BackgroundThe objectives of the study were to assess the knowledge, attitude, and practice of dentists towards providing oral health care to pregnant women and to identify barriers and predictors of periodontal and caries related perinatal oral healthcare practices.MethodsA cross-sectional analytical survey was conducted on dentists by using a random sampling technique, and a pre-validated questionnaire was delivered to 350 dentists from May 2018 to October 2018. Data were analyzed by utilizing SPSS software. Frequencies and percentages were recorded for descriptive variables. Binary logistic regression was used to analyze the probability of predicting group membership to the dependent variable using different independent variables determined from contingency tables.ResultsOverall response rate was 41%. The mean knowledge score of respondents was 15.86 ± 3.34. The lowest correct responses were noted in the questions related to periodontal health. It was found that the advice to delay dental visits until after pregnancy was eight times more likely to be observed among dentists who lacked the knowledge of importance of oral health during pregnancy (P = 0.04, OR = 8.75). Dentists were more likely to consult obstetricians regarding dental procedures when they fear a risk of labor in the dental practice (P < 0.05, OR = 3.72). Dentists who had the knowledge of periodontal disease association with preterm delivery were about four times more likely to treat periodontal disease during pregnancy (P = 0.01, OR = 3.95). Dentists knowing the association between maternal oral health and childhood decay were more likely to counsel pregnant patients regarding caries prevention (P > 0.05, OR = 3.75).ConclusionsCollectively the results indicated few gaps in knowledge among some dentists and a need to improve existing attitudes towards perinatal oral health. Dentists failing to recognize the importance of perinatal oral health are more likely to be hesitant in treating pregnant patients. Failing to recognize the link between periodontal disease and obstetric complications increases the possibility of hesitance to counsel pregnant patients regarding the same. The appreciation of the evidence for poor perinatal oral health and risk of early childhood caries increases the likelihood of counseling by dentists on caries prevention. creator: Muhammad Qasim Javed creator: Usman Anwer Bhatti creator: Arham Riaz creator: Farooq Ahmad Chaudhary uri: https://doi.org/10.7717/peerj.12080 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Javed et al. title: Towards a pragmatic use of statistics in ecology link: https://peerj.com/articles/12090 last-modified: 2021-09-01 description: Although null hypothesis testing (NHT) is the primary method for analyzing data in many natural sciences, it has been increasingly criticized. Recently, approaches based on information theory (IT) have become popular and were held by many to be superior because it enables researchers to properly assess the strength of the evidence that data provide for competing hypotheses. Many studies have compared IT and NHT in the context of model selection and stepwise regression, but a systematic comparison of the most basic uses of statistics by ecologists is still lacking. We used computer simulations to compare how both approaches perform in four basic test designs (t-test, ANOVA, correlation tests, and multiple linear regression). Performance was measured by the proportion of simulated samples for which each method provided the correct conclusion (power), the proportion of detected effects with a wrong sign (S-error), and the mean ratio of the estimated effect to the true effect (M-error). We also checked if the p-value from significance tests correlated to a measure of strength of evidence, the Akaike weight. In general both methods performed equally well. The concordance is explained by the monotonic relationship between p-values and evidence weights in simple designs, which agree with analytic results. Our results show that researchers can agree on the conclusions drawn from a data set even when they are using different statistical approaches. By focusing on the practical consequences of inferences, such a pragmatic view of statistics can promote insightful dialogue among researchers on how to find a common ground from different pieces of evidence. A less dogmatic view of statistical inference can also help to broaden the debate about the role of statistics in science to the entire path that leads from a research hypothesis to a statistical hypothesis. creator: Leonardo Braga Castilho creator: Paulo Inácio Prado uri: https://doi.org/10.7717/peerj.12090 license: https://creativecommons.org/licenses/by/4.0/ rights: © 2021 Castilho and Prado title: The bioinformatics and experimental analysis of AlkB family for prognosis and immune cell infiltration in hepatocellular carcinoma link: https://peerj.com/articles/12123 last-modified: 2021-09-01 description: BackgroundServing as N6-methyladenosine demethylases, the AlkB family is involved in the tumorigenesis of hepatocellular carcinoma (HCC). However, the molecular profiles and clinical values of the AlkB family in HCC are not well known.MethodsSeveral bioinformatics tools and in vitro experiments were used to identify the immune-related profiles and prognostic values of AlkB family in HCC.ResultsIn this study expression levels of ALKBH1/2/3/4/7 were all remarkably increased in HCC tissues when compared with normal tissues. Quantitative PCR (qPCR) and immunohistochemistry were used to validate the expression of AlkB family members in HCC tissues and normal liver tissues. In addition, high expression levels of ALKBH4 were negatively correlated with overall survival (OS) and disease-free survival (DFS) in patients with HCC. Increased ALKBH4 was also associated with pathological stage in HCC patients. The molecular profiles of AlkB family in HCC were mainly associated with peptidyl-serine modification, peptidyl-tyrosine modification, regulation of metal ion transport, etc. Furthermore, tumor-infiltrating immune cell analysis indicated that ALKBH1/2/3/4/5/6/7/8 and FTO were related to the infiltration of different immune cell, such as CD8+ T cells, macrophages, neutrophils, dendritic cells and CD4+ T cells. We also discovered that the methylation levels of ALKBH1/2/4/5/6/8 and FTO were remarkably reduced in HCC tissues.ConclusionsCollectively, our findings may deepen the understanding of specific molecular profiles of the AlkB family in HCC pathology. In particular, ALKBH4 could serve as a promising prognostic candidate for treating HCC, and these results might potentiate the development of more reliable therapeutic strategies for patients with HCC. creator: Bi Peng creator: Yuanliang Yan creator: Zhijie Xu uri: https://doi.org/10.7717/peerj.12123 license: https://creativecommons.org/licenses/by/4.0/ rights: ©2021 Peng et al.