title: PeerJ description: Articles published in PeerJ link: https://peerj.com/articles/index.rss3?journal=peerj&month=2019-04 creator: info@peerj.com PeerJ errorsTo: info@peerj.com PeerJ language: en title: Comparing imaging, acoustics, and radar to monitor Leach’s storm-petrel colonies link: https://peerj.com/articles/6721 last-modified: 2019-04-30 description: Seabirds are integral components of marine ecosystems and, with many populations globally threatened, there is a critical need for effective and scalable seabird monitoring strategies. Many seabird species nest in burrows, which can make traditional monitoring methods costly, infeasible, or damaging to nesting habitats. Traditional burrow occupancy surveys, where possible, can occur infrequently and therefore lead to an incomplete understanding of population trends. For example, in Oregon, during the last three decades there have been large changes in the abundance of Leach’s storm-petrels (Hydrobates leucorhoa), which included drastic declines at some colonies. Unfortunately, traditional monitoring failed to capture the timing and magnitude of change, limiting managers’ ability to determine causes of the decline and curtailing management options. New, easily repeatable methods of quantifying relative abundance are needed. For this study, we tested three methods of remote monitoring: passive acoustic monitoring, time-lapse cameras, and radar. Abundance indices derived from acoustics and imagery: call rates, acoustic energy, and counts were significantly related to traditional estimates of burrow occupancy of Leach’s storm-petrels. Due to sampling limitations, we were unable to compare radar to burrow occupancy. Image counts were significantly correlated with all other indices, including radar, while indices derived from acoustics and radar were not correlated. Acoustic data likely reflect different aspects of the population and hold the potential for the further development of indices to disentangle phenology, attendance of breeding birds, and reproductive success. We found that image counts are comparable with standard methods (e.g., radar) in producing annual abundance indices. We recommend that managers consider a sampling scheme that incorporates both acoustics and imaging, but for sites inaccessible to humans, radar remains the sole option. Implementation of acoustic and camera based monitoring programs will provide much needed information for a vulnerable group of seabirds. creator: Rachael A. Orben creator: Abram B. Fleishman creator: Abraham L. Borker creator: William Bridgeland creator: Amanda J. Gladics creator: Jessica Porquez creator: Peter Sanzenbacher creator: Shawn W. Stephensen creator: Roberta Swift creator: Matthew W. McKown creator: Robert M. Suryan uri: https://doi.org/10.7717/peerj.6721 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Orben et al. title: Clinical characteristics and prognosis of basaloid squamous cell carcinoma of the lung: a population-based analysis link: https://peerj.com/articles/6724 last-modified: 2019-04-30 description: BackgroundThis study analyzed the clinical features and prognosis of basaloid squamous cell carcinoma of the lung (BSC), and constructed a nomogram to predict the prognoses of patients.MethodsThe information of pure BSC patients was obtained in the Surveillance, Epidemiology, and End Results database between 2004 and 2015. Then, it was evaluated, and compared with the data of lung squamous cell carcinoma (SCC), lung large cell carcinoma (LCC) and lung adenocarcinoma (LAC) patients. Subsequently, we used univariate and multivariate analyses to investigate the independent factors related to the prognoses of patients with BSC and constructed a nomogram to verify the prognoses.ResultsA total of 425 patients diagnosed with BSC were enrolled. Compared with patients with SCC, LCC and LAC, the mean survival time of BSC patients was better than all of them. Compared with SCC, there were significant differences between the characteristics of grade (P < 0.001), total stage (P < 0.001), T stage (P < 0.001), N stage (P < 0.001), M stage (P < 0.001), surgery (P < 0.001), radiotherapy (P < 0.001), and chemotherapy (P < 0.001), while BSC also had significantly different clinical characteristics from LCC and LAC. Univariate and multivariate survival analyses showed that age (P < 0.001), T stage (P < 0.001), N stage (P = 0.009), M stage (P < 0.001), and surgery (P < 0.001) were independent prognostic factors of BSC. The survival of patients undergoing lobectomy was significantly better than sublobar resection, with an OR of 0.389 (0.263–0.578). We constructed a nomogram with a C-index of 0.750 (95% confidence interval) based on the results of multivariate analysis. The calibration curves based on nomogram scores indicated that the nomogram could accurately predict the prognosis of patients.ConclusionsBSC had unique clinical and prognostic features. T stage, N stage, M stage, age, and surgery were independently associated with overall survival (OS). Lobectomy was a relative ideal choice for patients with BSC. The nomogram effectively predicted the OS at 1-, 3-, and 5-years. creator: Guangda Yuan creator: Cheng Zhan creator: Yiwei Huang creator: Donglin Zhu creator: Hongya Xie creator: Tengteng Wei creator: Tao Lu creator: Qun Wang creator: Yong Yang creator: Yimeng Zhu uri: https://doi.org/10.7717/peerj.6724 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Yuan et al. title: Biodiversity seen through the perspective of insects: 10 simple rules on methodological choices and experimental design for genomic studies link: https://peerj.com/articles/6727 last-modified: 2019-04-30 description: Massively parallel DNA sequencing opens up opportunities for bridging multiple temporal and spatial dimensions in biodiversity research, thanks to its efficiency to recover millions of nucleotide polymorphisms. Here, we identify the current status, discuss the main challenges, and look into future perspectives on biodiversity genomics focusing on insects, which arguably constitute the most diverse and ecologically important group among all animals. We suggest 10 simple rules that provide a succinct step-by-step guide and best-practices to anyone interested in biodiversity research through the study of insect genomics. To this end, we review relevant literature on biodiversity and evolutionary research in the field of entomology. Our compilation is targeted at researchers and students who may not yet be specialists in entomology or molecular biology. We foresee that the genomic revolution and its application to the study of non-model insect lineages will represent a major leap to our understanding of insect diversity. creator: Pável Matos-Maraví creator: Camila Duarte Ritter creator: Christopher J. Barnes creator: Martin Nielsen creator: Urban Olsson creator: Niklas Wahlberg creator: Daniel Marquina creator: Ilari Sääksjärvi creator: Alexandre Antonelli uri: https://doi.org/10.7717/peerj.6727 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Matos-Maraví et al. title: Analysis of the spike, ORF3, and nucleocapsid genes of porcine epidemic diarrhea virus circulating on Thai swine farms, 2011–2016 link: https://peerj.com/articles/6843 last-modified: 2019-04-30 description: Porcine epidemic diarrhea virus (PEDV) outbreaks on pig farms have caused significant economic loss in the swine industry since it was first reported in Thailand a decade ago. Anecdotal evidence suggests that PEDV is now endemic in this region, therefore genome information of circulating PEDV is important for molecular surveillance and evaluation of potential benefits of field vaccination. Here, we characterized PEDV infection on commercial Thai swine farms by screening 769 samples of feces and small intestinal contents from pigs with diarrhea between 2011 and 2016. Using reverse-transcription polymerase chain reaction targeting the spike (S) gene, 153 PEDV-positive samples were further subjected to analysis of the open reading frame 3 and nucleocapsid (N) genes. Comparison of 95 samples in which nucleotide sequencing was successfully obtained for all three genes revealed evolutionary diversity among the Thai PEDV strains. Phylogenetic analyses suggest that although some Thai strains changed little from years past, others resembled more closely to the recent strains reported in China. Interestingly, eight Thai PEDV strains possessed amino acid deletions in the N protein. The PEDV sequence divergence may be responsible for driving periodic outbreaks and continued persistence of PEDV on commercial swine farms. Our findings provide important insight into regional PEDV strains in circulation, which may assist future inclusions of suitable strains for future PEDV vaccines. creator: Supansa Tuanthap creator: Sompong Vongpunsawad creator: Cherdpong Phupolphan creator: Ausanee Duang-in creator: Suphot Wattanaphansak creator: Pornchalit Assavacheep creator: Apiradee Theamboonlers creator: Supol Luengyosluechakul creator: Alongkorn Amonsin creator: Yong Poovorawan uri: https://doi.org/10.7717/peerj.6843 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Tuanthap et al. title: Captivity causes taxonomic and functional convergence of gut microbial communities in bats link: https://peerj.com/articles/6844 last-modified: 2019-04-30 description: BackgroundDiet plays a crucial role in sculpting microbial communities. Similar diets appear to drive convergence of gut microbial communities between host species. Captivity usually provides an identical diet and environment to different animal species that normally have similar diets. Whether different species’ microbial gut communities can be homogenized by a uniform diet in captivity remains unclear.MethodsIn this study, we compared gut microbial communities of three insectivorous bat species (Rhinolophus ferrumequinum, Vespertilio sinensis, and Hipposideros armiger) in captivity and in the wild using 16S rDNA sequencing. In captivity, R. ferrumequinum and V. sinensis were fed yellow mealworms, while H. armiger was fed giant mealworms to rule out the impact of an identical environment on the species’ gut microbial communities.ResultsWe found that the microbial communities of the bat species we studied clustered by species in the wild, while the microbial communities of R. ferrumequinum and V. sinensis in captivity clustered together. All microbial functions found in captive V. sinensis were shared by R. ferrumequinum. Moreover, the relative abundances of all metabolism related KEGG pathways did not significantly differ between captive R. ferrumequinum and V. sinensis; however, the relative abundance of “Glycan Biosynthesis and Metabolism” differed significantly between wild R. ferrumequinum and V. sinensis.ConclusionOur results suggest that consuming identical diets while in captivity tends to homogenize the gut microbial communities among bat species. This study further highlights the importance of diet in shaping animal gut microbiotas. creator: Yanhong Xiao creator: Guohong Xiao creator: Heng Liu creator: Xin Zhao creator: Congnan Sun creator: Xiao Tan creator: Keping Sun creator: Sen Liu creator: Jiang Feng uri: https://doi.org/10.7717/peerj.6844 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Xiao et al. title: Exploring the transcriptome of luxI− and ΔainS mutants and the impact of N-3-oxo-hexanoyl-L- and N-3-hydroxy-decanoyl-L-homoserine lactones on biofilm formation in Aliivibrio salmonicida link: https://peerj.com/articles/6845 last-modified: 2019-04-30 description: BackgroundBacterial communication through quorum sensing (QS) systems has been reported to be important in coordinating several traits such as biofilm formation. In Aliivibrio salmonicida two QS systems the LuxI/R and AinS/R, have been shown to be responsible for the production of eight acyl-homoserine lactones (AHLs) in a cell density dependent manner. We have previously demonstrated that inactivation of LitR, the master regulator of the QS system resulted in biofilm formation, similar to the biofilm formed by the AHL deficient mutant ΔainSluxI−. In this study, we aimed to investigate the global gene expression patterns of luxI and ainS autoinducer synthases mutants using transcriptomic profiling. In addition, we examined the influence of the different AHLs on biofilm formation.ResultsThe transcriptome profiling of ΔainS and luxI− mutants allowed us to identify genes and gene clusters regulated by QS in A. salmonicida. Relative to the wild type, the ΔainS and luxI− mutants revealed 29 and 500 differentially expressed genes (DEGs), respectively. The functional analysis demonstrated that the most pronounced DEGs were involved in bacterial motility and chemotaxis, exopolysaccharide production, and surface structures related to adhesion. Inactivation of luxI, but not ainS genes resulted in wrinkled colony morphology. While inactivation of both genes (ΔainSluxI−) resulted in strains able to form wrinkled colonies and mushroom structured biofilm. Moreover, when the ΔainSluxI− mutant was supplemented with N-3-oxo-hexanoyl-L-homoserine lactone (3OC6-HSL) or N-3-hydroxy-decanoyl-L-homoserine lactone (3OHC10-HSL), the biofilm did not develop. We also show that LuxI is needed for motility and for repression of EPS production, where repression of EPS is likely operated through the RpoQ-sigma factor.ConclusionThese findings imply that the LuxI and AinS autoinducer synthases play a critical role in the regulation of biofilm formation, EPS production, and motility. creator: Miriam Khider creator: Hilde Hansen creator: Erik Hjerde creator: Jostein A. Johansen creator: Nils Peder Willassen uri: https://doi.org/10.7717/peerj.6845 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Khider et al. title: Identification and comparative analysis of the CIPK gene family and characterization of the cold stress response in the woody plant Prunus mume link: https://peerj.com/articles/6847 last-modified: 2019-04-30 description: Prunus mume is an important ornamental woody plant that grows in tropical and subtropical regions. Freezing stress can adversely impact plant productivity and limit the expansion of geographical locations. Understanding cold-responsive genes could potentially bring about the development of new ways to enhance plant freezing tolerance. Members of the serine/threonine protein kinase (CIPK) gene family play important roles in abiotic stress. However, the function of CIPK genes in P. mume remains poorly defined. A total of 16 CIPK genes were first identified in P. mume. A systematic phylogenetic analysis was conducted in which 253 CIPK genes from 12 species were divided into three groups. Furthermore, we analysed the chromosomal locations, molecular structures, motifs and domains of CIPK genes in P. mume. All of the CIPK sequences had NAF domains and promoter regions containing cis-acting regulatory elements of the related stress response. Three PmCIPK genes were identified as Pmu-miR172/167-targeted sites. Transcriptome data showed that most PmCIPK genes presented tissue-specific and time-specific expression profiles. Nine genes were highly expressed in flower buds in December and January, and 12 genes were up-regulated in stems in winter. The expression levels of 12 PmCIPK genes were up-regulated during cold stress treatment confirmed by qRT-PCR. Our study improves understanding of the role of the PmCIPK gene family in the low temperature response in woody plants and provides key candidate genes and a theoretical basis for cold resistance molecular-assisted breeding technology in P. mume. creator: Ping Li creator: Tangchun Zheng creator: Lulu Li creator: Xiaokang Zhuo creator: Liangbao Jiang creator: Jia Wang creator: Tangren Cheng creator: Qixiang Zhang uri: https://doi.org/10.7717/peerj.6847 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Li et al. title: Quality of life of older Chinese adults receiving primary care in Wuhan, China: a multi-center study link: https://peerj.com/articles/6860 last-modified: 2019-04-30 description: BackgroundQuality of life (QOL) is an important primary care outcome, but the QOL of older adults treated in primary care is understudied in China. This study examined QOL and its associated factors in older adults treated in Chinese primary care.MethodsA total of 752 older patients (65+ years) were consecutively recruited from 13 primary care centers in Wuhan, China, and interviewed with a standardized questionnaire, concerning socio-demographics, major medical conditions, loneliness, and depression. QOL and depression were measured with the Chinese six-item QOL questionnaire and the shortened Geriatric Depression Scale, respectively. Multiple linear regression was used to identify factors associated with poor QOL.ResultsThe average QOL score of primary care older adults was (20.7 ± 2.5), significantly lower than that of the Chinese general population. Factors significantly associated with poor QOL of Chinese primary care older adults included engaging in manual labor before older adulthood (unstandardized coefficient [β]: −0.702, P < 0.001), no living adult children (β: −1.720, P = 0.001), physical inactivity (β: −0.696, P < 0.001), having ≥ four major medical conditions (β: −1.813, P < 0.001), hearing problem (β: −1.004, P = 0.017), depression (β: −1.153, P < 0.001), and loneliness (β: −1.396, P < 0.001).ConclusionsOlder adults treated in Chinese primary care have poorer QOL than the general population. Addressing psychosocial problems at Chinese primary care settings could be helpful in improving QOL in Chinese older adults. creator: Bao-Liang Zhong creator: Yan-Min Xu creator: Wu-Xiang Xie creator: Xiu-Jun Liu uri: https://doi.org/10.7717/peerj.6860 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Zhong et al. title: Development of enhancer-trapping and -detection vectors mediated by the Tol2 transposon in zebrafish link: https://peerj.com/articles/6862 last-modified: 2019-04-30 description: Enhancers are key transcriptional drivers of gene expression. The identification of enhancers in the genome is central for understanding gene-expression programs. Although transposon-mediated enhancer trapping (ET) is a powerful approach to the identification of enhancers in zebrafish, its efficiency varies considerably. To improve the ET efficiency, we constructed Tol2-mediated ET vectors with a reporter gene (mCherry) expression box driven by four minimal promoters (Gata, Myc, Krt4 and Oct4), respectively. The ET efficiency and expression background were compared among the four promoters by zebrafish embryo injection at the one-cell stage. The results showed that the Gata minimal promoter yielded the lowest basic expression and the second-highest trapping efficiency (44.6% at 12 hpf (hour post-fertilization) and 23.1% at 72 hpf, n = 305 and n = 307). The Krt4 promoter had the highest trapping efficiency (64% at 12 hpf and 67.1% at 72 hpf, n = 302 and n = 301) and the strongest basic expression. To detect enhancer activity, chicken 5′HS4 double insulators were cloned into the two ET vectors with the Gata or Krt4 minimal promoter, flanking the mCherry expression box. The resulting detection vectors were injected into zebrafish embryos. mCherry expression driven by the Gata promoter (about 5%, n = 301) was decreased significantly compared with that observed for embryos injected with the ET vectors (23% at 72 hpf, n = 308). These results suggest that the insulators block the genome-position effects and that this vector is fit for enhancer-activity evaluation. To assess the compatibility between the enhancers and the minimal promoters, four enhancers (CNS1, Z48, Hand2 and Hs769) were cloned upstream of the Gata or Beta-globin minimal promoter in the enhancer-activity-detection vectors. The resulting recombinant vectors were assayed by zebrafish embryo injection. We found that Z48 and CNS1 responded to the Gata minimal promoter, and that Hand2 only responded to the Beta-globin minimal promoter. In contrast, Hs769 did not respond to either the Gata or Beta-globin minimal promoters. These results suggest the existence of compatibility between enhancers and minimal promoters. This study represents a systematic approach to the discovery of optional ET and enhancer-detection vectors. We are eager to provide a superior tool for understanding functional genomics. creator: Shuheng Chan creator: Dan Shen creator: Yatong Sang creator: Saisai Wang creator: Yali Wang creator: Cai Chen creator: Bo Gao creator: Chengyi Song uri: https://doi.org/10.7717/peerj.6862 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Chan et al. title: A citizen science approach to evaluating US cities for biotic homogenization link: https://peerj.com/articles/6879 last-modified: 2019-04-30 description: Cities around the world have converged on structural and environmental characteristics that exert similar eco-evolutionary pressures on local communities. However, evaluating how urban biodiversity responds to urban intensification remains poorly understood because of the challenges in capturing the diversity of a range of taxa within and across multiple cities from different types of urbanization. Here we utilize a growing resource—citizen science data. We analyzed 66,209 observations representing 5,209 species generated by the City Nature Challenge project on the iNaturalist platform, in conjunction with remote sensing (NLCD2011) environmental data, to test for urban biotic homogenization at increasing levels of urban intensity across 14 metropolitan cities in the United States. Based on community composition analyses, we found that while similarities occur to an extent, urban biodiversity is often much more a reflection of the taxa living locally in a region. At the same time, the communities found in high-intensity development were less explained by regional context than communities from other land cover types were. We also found that the most commonly observed species are often shared between cities and are non-endemic and/or have a distribution facilitated by humans. This study highlights the value of citizen science data in answering questions in urban ecology. creator: Misha Leong creator: Michelle Trautwein uri: https://doi.org/10.7717/peerj.6879 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Leong and Trautwein title: Genetic differentiation in the southern population of the Fathead Minnow Pimephales promelas Rafinesque (Actinopterygii: Cyprinidae) link: https://peerj.com/articles/6224 last-modified: 2019-04-29 description: The North American cyprinid Pimephales promelas is a species with a wide distribution range, occurring in distinct hydrographic basins in Mexico, Canada, and the United States. Previous morphological and meristic analyses of P. promelas concluded that at least three subspecies exist in the midwestern and northeast region of the United States. No studies have been carried out on the Mexican population of P. promelas, but the findings of cryptic diversity in United States populations of this species, as well as in other codistributed fish species in Mexico could be an indication that Mexican populations of P. promelas consist of cryptic species. Using the mitochondrial gene cyt b and the first intron of the S7 ribosomal protein-coding nuclear gene we carried out phylogenetic and phylogeographic analyses of populations of P. promelas across its distribution range in northwestern Mexico. Using this information were analyzed the structure and differentiation level between populations of P. promelas from distinct river basins in the region in identifying cryptic diversity. Twenty-four sequences were obtained for cyt b, and 30 for S7, which included the two heterozygous alleles. The results revealed the existence of four well-differentiated lineages: (1) Yaqui in the Pacific slope; (2) Santa Maria, and (3) Casas Grandes in the Guzman Basin; and (4) Nazas+Conchos in Chihuahua state. This challenges the current taxonomy of P. promelas. Differences in the relationships between markers and the small sample size for the Santa Maria population (n = 1), indicate that our results must be corroborated with more data and morphological analyses. Biogeographic analysis of these findings suggest that the evolutionary history of P. promelas is associated with the fragmentation of the ancestral Rio Grande river system since Miocene in northwestern Mexico consistent with findings for codistributed fish species. creator: Nayarit E. Ballesteros-Nova creator: Rodolfo Pérez-Rodríguez creator: Rosa G. Beltrán-López creator: Omar Domínguez-Domínguez uri: https://doi.org/10.7717/peerj.6224 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Ballesteros-Nova et al. title: A prevalence of Arthropterygius (Ichthyosauria: Ophthalmosauridae) in the Late Jurassic—earliest Cretaceous of the Boreal Realm link: https://peerj.com/articles/6799 last-modified: 2019-04-29 description: The ichthyosaur genus ArthropterygiusMaxwell, 2010 is considered as rare and poorly known. However, considering the existing uncertainty regarding its position in respect to ophthalmosaurid subfamilies in recent phylogenies, it is among the key taxa for understanding the evolution of derived Late Jurassic and Early Cretaceous ichthyosaurs. Recently excavated unique material from the Berriassian of Franz Josef Land (Russian Extreme North) and examination of historical collections in Russian museums provided numerous specimens referable to Arthropterygius. The new data combined with personal examination of ichthyosaurs Palvennia, Janusaurus, and Keilhauia from Svalbard give us reason to refer all these taxa to Arthropterygius. Therefore, we recognize four species within the genus: Arthropterigius chrisorum (Russell, 1994), A. volgensis (Kasansky, 1903) comb. nov., A. hoybergeti (Druckenmiller et al., 2012) comb. nov., and A. lundi (Roberts et al., 2014) comb. nov. Three of the species are found both in the Arctic and in the European Russia. This allows the suggestion that Arthropterygius was common and widespread in the Boreal Realm during the Late Jurassic and earliest Cretaceous. The results of our multivariate analysis of ophthalmosaurid humeral morphology indicate that at least some ophthalmosaurid genera and species, including Arthropterygius, could be easily recognized based solely on humeral morphology. Our phylogenetic analyses place the clade of Arthropterygius close to the base of Ophthalmosauria as a sister group either to ophthalmosaurines or to platypterygiines. Although its position is still uncertain, this is the best supported clade of ophthalmosaurids (Bremer support value of 5, Bootstrap and Jackknife values exceeding 80) that further augments our taxonomic decision. creator: Nikolay G. Zverkov creator: Natalya E. Prilepskaya uri: https://doi.org/10.7717/peerj.6799 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Zverkov and Prilepskaya title: Integrated analysis reveals five potential ceRNA biomarkers in human lung adenocarcinoma link: https://peerj.com/articles/6694 last-modified: 2019-04-29 description: BackgroundCompeting endogenous RNAs (ceRNAs) are a newly identified type of regulatory RNA. Accumulating evidence suggests that ceRNAs play an important role in the pathogenesis of diseases such as cancer. Thus, ceRNA dysregulation may represent an important molecular mechanism underlying cancer progression and poor prognosis. In this study, we aimed to identify ceRNAs that may serve as potential biomarkers for early diagnosis of lung adenocarcinoma (LUAD).MethodsWe performed differential gene expression analysis on TCGA-LUAD datasets to identify differentially expressed (DE) mRNAs, lncRNAs, and miRNAs at different tumor stages. Based on the ceRNA hypothesis and considering the synergistic or feedback regulation of ceRNAs, a lncRNA–miRNA–mRNA network was constructed. Functional analysis was performed using gene ontology term and KEGG pathway enrichment analysis and KOBAS 2.0 software. Transcription factor (TF) analysis was carried out to identify direct targets of the TFs associated with LUAD prognosis. Identified DE genes were validated using gene expression omnibus (GEO) datasets.ResultsBased on analysis of TCGA-LUAD datasets, we obtained 2,610 DE mRNAs, 915 lncRNAs, and 125 miRNAs that were common to different tumor stages (|log2(Fold change)| ≥ 1, false discovery rate < 0.01), respectively. Functional analysis showed that the aberrantly expressed mRNAs were closely related to tumor development. Survival analyses of the constructed ceRNA network modules demonstrated that five of them exhibit prognostic significance. The five ceRNA interaction modules contained one lncRNA (FENDRR), three mRNAs (EPAS1, FOXF1, and EDNRB), and four miRNAs (hsa-miR-148a, hsa-miR-195, hsa-miR-196b, and hsa-miR-301b). The aberrant expression of one lncRNA and three mRNAs was verified in the LUAD GEO dataset. Transcription factor analysis demonstrated that EPAS1 directly targeted 13 DE mRNAs.ConclusionOur observations indicate that lncRNA-related ceRNAs and TFs play an important role in LUAD. The present study provides novel insights into the molecular mechanisms underlying LUAD pathogenesis. Furthermore, our study facilitates the identification of potential biomarkers for the early diagnosis and prognosis of LUAD and therapeutic targets for its treatment. creator: Yu Liu creator: Deyao Xie creator: Zhifeng He creator: Liangcheng Zheng uri: https://doi.org/10.7717/peerj.6694 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Liu et al. title: Shared genomic outliers across two divergent population clusters of a highly threatened seagrass link: https://peerj.com/articles/6806 last-modified: 2019-04-29 description: The seagrass, Zostera capensis, occurs across a broad stretch of coastline and wide environmental gradients in estuaries and sheltered bays in southern and eastern Africa. Throughout its distribution, habitats are highly threatened and poorly protected, increasing the urgency of assessing the genomic variability of this keystone species. A pooled genomic approach was employed to obtain SNP data and examine neutral genomic variation and to identify potential outlier loci to assess differentiation across 12 populations across the ∼9,600 km distribution of Z. capensis. Results indicate high clonality and low genomic diversity within meadows, which combined with poor protection throughout its range, increases the vulnerability of this seagrass to further declines or local extinction. Shared variation at outlier loci potentially indicates local adaptation to temperature and precipitation gradients, with Isolation-by-Environment significantly contributing towards shaping spatial variation in Z. capensis. Our results indicate the presence of two population clusters, broadly corresponding to populations on the west and east coasts, with the two lineages shaped only by frequency differences of outlier loci. Notably, ensemble modelling of suitable seagrass habitat provides evidence that the clusters are linked to historical climate refugia around the Last Glacial Maxi-mum. Our work suggests a complex evolutionary history of Z. capensis in southern and eastern Africa that will require more effective protection in order to safeguard this important ecosystem engineer into the future. creator: Nikki Leanne Phair creator: Robert John Toonen creator: Ingrid Knapp creator: Sophie von der Heyden uri: https://doi.org/10.7717/peerj.6806 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Phair et al. title: Seed survival of Australian Acacia in the Western Cape of South Africa in the presence of biological control agents and given environmental variation link: https://peerj.com/articles/6816 last-modified: 2019-04-29 description: Studies of invasive Australian Acacia have shown that many seeds are still produced and accumulate in soil stored seed banks regardless of the presence of seed-targeting biological control agents. This is despite claims of biological control success, although there is generally a lack of data on the seed production of invasive Australian Acacia before and after the release of the respective agents. We aimed to quantify seed production and seed survival of invasive Australian Acacia currently under biological control. The seed production and survival (proportion of aborted, predated and surviving seeds) of A. longifolia, A. pycnantha and A. saligna were each studied at four to five sites in the Western Cape of South Africa. The relationships between seed production and stand characteristics were determined and the relative effects of seed predation and abortion on seed survival were established. The investigated invasive Australian Acacia produced many seeds that survived the pre-dispersal stage despite long-term presence of released biological control agents. It was shown that seed crop size is the only significant factor influencing seed survival of the studied Australian Acacia species. Furthermore, the seeds surviving per tree and per square meter were related to tree size. No quantitative evidence was found to suggest that seed-reducing biological control agents are having an impact on the population dynamics of their Australian Acacia hosts. This study illustrates the importance of studying the seed ecology of invasive plants before biological control agents are selected and released. creator: Matthys Strydom creator: Ruan Veldtman creator: Mzabalazo Z. Ngwenya creator: Karen J. Esler uri: https://doi.org/10.7717/peerj.6816 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Strydom et al. title: Prolonged pituitary down-regulation with full-dose of gonadotropin-releasing hormone agonist in different menstrual cycles: a retrospective cohort study link: https://peerj.com/articles/6837 last-modified: 2019-04-29 description: BackgroundThe efficiency of prolonged down-regulation caused by a full-dose of gonadotropin-releasing hormone agonist (GnRH-a) injected during different menstrual phases has not yet been researched. Our goal was to evaluate the effects of GnRH-a, which was used in different phases of the menstrual cycle in patients undergoing in vitro fertilization and embryo transfer.MethodsThis was a retrospective cohort study. A total of 320 patients received a prolonged pituitary down-regulated full-dose (3.75 mg) of triptorelin in the early follicular phase, and 160 patients received the same full-dose of triptorelin during the mid-luteal phase. Clinical and laboratory outcomes were compared between the two groups.ResultsThe basic characteristics of the two groups were comparable. The mean number of retrieved oocytes, fertilized oocytes, cleavage oocytes and good quality embryos were comparable between the two groups. Although there was a higher antral follicle count, cyst formation rate, fertilization rate and cleavage rate in the follicular phase group, no statistically significant effects were seen on implantation rate (41.15% vs. 45.91%), clinical pregnancy rate (60.38% vs. 61.36%), ongoing pregnancy rate (57.74% vs. 57.58%), live birth rate (56.23% vs. 57.58%) or early abortion rate (2.64% vs. 3.79%) per fresh transfer cycle. Moreover, severe ovarian hyperstimulation syndrome rates at the early stage (1.89% vs. 2.27%) were low in both groups.ConclusionsProlonged pituitary down-regulation achieved by utilizing a full-dose of GnRH-a administrated in either phase of the menstrual cycle can have a positive effect on ongoing pregnancy rate and live-birth rate per fresh embryo transfer cycle. Ovarian cyst formation rate was higher in the follicular phase group, but this did not have any adverse impact on clinical results. creator: Yingfen Ying creator: Tanchu Yang creator: Huina Zhang creator: Chang Liu creator: Junzhao Zhao uri: https://doi.org/10.7717/peerj.6837 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Ying et al. title: Maintaining fixation does not increase demands on working memory relative to free viewing link: https://peerj.com/articles/6839 last-modified: 2019-04-29 description: The comparison of memory performance during free and fixed viewing conditions has been used to demonstrate the involvement of eye movements in memory encoding and retrieval, with stronger effects at encoding than retrieval. Relative to conditions of free viewing, participants generally show reduced memory performance following sustained fixation, suggesting that unrestricted eye movements benefit memory. However, the cognitive basis of the memory reduction during fixed viewing is uncertain, with possible mechanisms including disruption of visual-mnemonic and/or imagery processes with sustained fixation, or greater working memory demands required for fixed relative to free viewing. To investigate one possible mechanism for this reduction, we had participants perform a working memory task—an auditory n-back task—during free and fixed viewing, as well as a repetitive finger tapping condition, included to isolate the effects of motor interference independent of the oculomotor system. As expected, finger tapping significantly interfered with n-back performance relative to free viewing, as indexed by a decrease in accuracy and increase in response times. By contrast, there was no evidence that fixed viewing interfered with n-back performance relative to free viewing. Our findings failed to support a hypothesis of increased working memory load during fixation. They are consistent with the notion that fixation disrupts long-term memory performance through interference with visual processes. creator: Michael J. Armson creator: Jennifer D. Ryan creator: Brian Levine uri: https://doi.org/10.7717/peerj.6839 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Armson et al. title: Effects of thermal stress on amount, composition, and antibacterial properties of coral mucus link: https://peerj.com/articles/6849 last-modified: 2019-04-29 description: The surface mucus layer of reef-building corals supports feeding, sediment clearing, and protection from pathogenic invaders. As much as half of the fixed carbon supplied by the corals’ photosynthetic symbionts is incorporated into expelled mucus. It is therefore reasonable to expect that coral bleaching (disruption of the coral–algal symbiosis) would affect mucus production. Since coral mucus serves as an important nutrient source for the entire reef community, this could have substantial ecosystem-wide consequences. In this study, we examined the effects of heat stress-induced coral bleaching on the composition and antibacterial properties of coral mucus. In a controlled laboratory thermal challenge, stressed corals produced mucus with higher protein (β = 2.1, p < 0.001) and lipid content (β = 15.7, p = 0.02) and increased antibacterial activity (likelihood ratio = 100, p < 0.001) relative to clonal controls. These results are likely explained by the expelled symbionts in the mucus of bleached individuals. Our study suggests that coral bleaching could immediately impact the nutrient flux in the coral reef ecosystem via its effect on coral mucus. creator: Rachel M. Wright creator: Marie E. Strader creator: Heather M. Genuise creator: Mikhail Matz uri: https://doi.org/10.7717/peerj.6849 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Wright et al. title: A combination of curcumin, vorinostat and silibinin reverses Aβ-induced nerve cell toxicity via activation of AKT-MDM2-p53 pathway link: https://peerj.com/articles/6716 last-modified: 2019-04-26 description: Alzheimer’s disease (AD) is a significant health issue for the elderly and becoming increasingly common as the global population ages. Although many efforts have been made to elucidate its pathology, there is still a lack of effective clinical anti-AD agents. Previous research has shown the neuroprotective properties of a combination of curcumin and vorinostat. In this study, nine other neuroprotective agents were investigated to examine whether a three-drug combination of curcumin, vorinostat, and a new drug is more advantageous than the previous two-drug combination in alleviating amyloid beta (Aβ)-induced nerve cell toxicity. Cell viability assay was performed to screen these agents, and further validation tests, including determination of cellular oxidative stress, apoptosis, and activity of the AKT/MDM2/p53 pathway, were performed. Among the nine candidate compounds, only silibinin at 1 µM reduced Aβ25–35-induced toxicity in PC12 cells. The neuroprotective effects of 1 µM silibinin in combination with 5 µM curcumin and 0.5 µM vorinostat (CVS) was shown in PC12 cells, in which it decreased apoptosis and oxidative stress marker levels that were increased by 20 µM Aβ25–35. Western blotting results showed that CVS pretreatment significantly increased the phosphorylation of AKT, BAD, and MDM2, which resulted in decreased intracellular expression of p53. Further, immunofluorescence results showed reduced p53 levels in the nuclei of PC12 cells following CVS pretreatment, indicating a reduction in the p53-mediated transcriptional activity associated with Aβ25–35 exposure. In conclusion, our findings suggested that pretreatment with CVS protected PC12 cells from Aβ25–35-induced toxicity through modulation of the AKT/MDM2/p53 pathway. Thus, CVS may present a new therapeutic option for treating AD. creator: Jia Meng creator: Yan Li creator: Mingming Zhang creator: Wenjing Li creator: Lin Zhou creator: Qiujun Wang creator: Lin Lin creator: Lihong Jiang creator: Wenliang Zhu uri: https://doi.org/10.7717/peerj.6716 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Meng et al. title: Impact of emotional and motivational regulation on putting performance: a frontal alpha asymmetry study link: https://peerj.com/articles/6777 last-modified: 2019-04-26 description: BackgroundThe efficacy of emotional and motivational regulation can determine athletic performance. Giving the short duration and fast changing nature of emotions experienced by athletes in competition, it is important to examine the temporal dynamics of emotional and motivational regulation. The aim of this study was to investigate emotional and motivational regulation as measured by frontal alpha asymmetry in skilled golfers during putting performance after a performance failure.MethodsTwenty skilled university golfers were recruited and requested to perform 40 putts at an individualized difficulty level of 40–60% successful putting rate. Trials immediately after a failed putt were selected for analysis. Successful performances were those trials where a hole was and unsuccessful performances were those that failed. The frontal alpha asymmetry index of LnF4-LnF3 was derived for statistical analysis.Results(1) Successful performance was preceded by a larger frontal alpha asymmetry index at T2 than that of T1, and (2) a larger frontal alpha asymmetry index was observed for unsuccessful performance than for successful performance at T1.DiscussionThe results suggest that successful emotional and motivational regulation was characterized by a progressive increase of frontal alpha asymmetry, which led to subsequent putting success when facing an emotionally provocative putting failure. These findings shed light on the application of frontal alpha asymmetry for the understanding and enhancement of emotional and motivational regulation during sport performance. creator: Tai-Ting Chen creator: Kuo-Pin Wang creator: Ming-Yang Cheng creator: Yi-Ting Chang creator: Chung-Ju Huang creator: Tsung-Min Hung uri: https://doi.org/10.7717/peerj.6777 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Chen et al. title: OsteoporosAtlas: a human osteoporosis-related gene database link: https://peerj.com/articles/6778 last-modified: 2019-04-26 description: BackgroundOsteoporosis is a common, complex disease of bone with a strong heritable component, characterized by low bone mineral density, microarchitectural deterioration of bone tissue and an increased risk of fracture. Due to limited drug selection for osteoporosis and increasing morbidity, mortality of osteoporotic fractures, osteoporosis has become a major health burden in aging societies. Current researches for identifying specific loci or genes involved in osteoporosis contribute to a greater understanding of the pathogenesis of osteoporosis and the development of better diagnosis, prevention and treatment strategies. However, little is known about how most causal genes work and interact to influence osteoporosis. Therefore, it is greatly significant to collect and analyze the studies involved in osteoporosis-related genes. Unfortunately, the information about all these osteoporosis-related genes is scattered in a large amount of extensive literature. Currently, there is no specialized database for easily accessing relevant information about osteoporosis-related genes and miRNAs.MethodsWe extracted data from literature abstracts in PubMed by text-mining and manual curation. Moreover, a local MySQL database containing all the data was developed with PHP on a Windows server.ResultsOsteoporosAtlas (http://biokb.ncpsb.org/osteoporosis/), the first specialized database for easily accessing relevant information such as osteoporosis-related genes and miRNAs, was constructed and served for researchers. OsteoporosAtlas enables users to retrieve, browse and download osteoporosis-related genes and miRNAs. Gene ontology and pathway analyses were integrated into OsteoporosAtlas. It currently includes 617 human encoding genes, 131 human non-coding miRNAs, and 128 functional roles. We think that OsteoporosAtlas will be an important bioinformatics resource to facilitate a better understanding of the pathogenesis of osteoporosis and developing better diagnosis, prevention and treatment strategies. creator: Xun Wang creator: Lihong Diao creator: Dezhi Sun creator: Dan Wang creator: Jiarun Zhu creator: Yangzhige He creator: Yuan Liu creator: Hao Xu creator: Yi Zhang creator: Jinying Liu creator: Yan Wang creator: Fuchu He creator: Yang Li creator: Dong Li uri: https://doi.org/10.7717/peerj.6778 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Wang et al. title: MorphoCatcher: a multiple-alignment based web tool for target selection and designing taxon-specific primers in the loop-mediated isothermal amplification method link: https://peerj.com/articles/6801 last-modified: 2019-04-26 description: BackgroundAdvantages of loop-mediated isothermal amplification in molecular diagnostics allow to consider the method as a promising technology of nucleic acid detection in agriculture and medicine. A bioinformatics tool that provides rapid screening and selection of target nucleotide sequences with subsequent taxon-specific primer design toward polymorphic orthologous genes, not only unique or conserved common regions of genome, would contribute to the development of more specific and sensitive diagnostic assays. However, considering features of the original software for primer selection, also known as the PrimerExplorer (Eiken Chemical Co. LTD, Tokyo, Japan), the taxon-specific primer design using multiple sequence alignments of orthologs or even viral genomes with conservative architecture is still complicated.FindingsHere, MorphoCatcher is introduced as a fast and simple web plugin for PrimerExplorer with a clear interface. It enables an execution of multiple-alignment based search of taxon-specific mutations, visual screening and selection of target sequences, and easy-to-start specific primer design using the PrimerExplorer software. The combination of MorphoCatcher and PrimerExplorer allows to perform processing of the multiple alignments of orthologs for informative sliding-window plot analysis, which is used to identify the sequence regions with a high density of taxon-specific mutations and cover them by the primer ends for better specificity of amplification.ConclusionsWe hope that this new bioinformatics tool developed for target selection and taxon-specific primer design, called the MorphoCatcher, will gain more popularity of the loop-mediated isothermal amplification method for molecular diagnostics community. MorphoCatcher is a simple web plugin tool for the PrimerExplorer software which is freely available only for non-commercial and academic users at http://morphocatcher.ru. creator: Fedor V. Shirshikov creator: Yuri A. Pekov creator: Konstantin A. Miroshnikov uri: https://doi.org/10.7717/peerj.6801 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Shirshikov et al. title: Risk factors for repeat percutaneous coronary intervention in young patients (≤45 years of age) with acute coronary syndrome link: https://peerj.com/articles/6804 last-modified: 2019-04-26 description: BackgroundThe incidences of premature coronary heart disease present a rising trend worldwide. The possible risk factors that may predict the incidence of repeat percutaneous coronary intervention (PCI) in premature acute coronary syndrome (ACS) remains unclear.MethodsA total of 203 patients ≤45 years with ACS from Chinese PLA General Hospital who have undergone angiography twice were included in this report. Data were collected from medical records of patients during hospitalization. Baseline characteristics which have significant differences in the univariate analysis were enrolled into the multiple logistic regression analysis. According to the odds ratio (OR) of these variables, different values were assigned to build a risk model to predict the possible risk of the premature ACS patients undergoing repeat PCI.ResultsOf the 203 young patients, 88 patients (43.3%) underwent repeat PCI. The intermit time (OR 1.002, (95% CI [1.001–1.002])), diastolic blood pressure of second procedure (OR 0.967, (95% CI [0.938–0.996])), stent diameter (OR 0.352, (95% CI [0.148–0.840])), HbA1C of the first procedure (OR 1.835, (95% CI [1.358–2.479])), and Troponin T of the second procedure (OR 1.24, (95% CI [0.981–1.489])) were significantly associated with the incidence of repeat PCI in patients with premature ACS. An aggregate score between 0 and 6 was calculated based on these cutpoints.ConclusionFor young patients with premature ACS, risk of undergoing repeat PCI was high. HbA1C was a significant, independent predictor for the incidence of repeat revascularization, and weighed more than traditional lipid profile. The glucose metabolism and disorders in patients with premature ACS should be routinely screened. creator: Tianwen Han creator: Qun Wang creator: Huanwan Yang creator: Shanshan Zhou creator: Jing Wang creator: Jing Jing creator: Tao Zhang creator: Yuqi Liu creator: Yundai Chen uri: https://doi.org/10.7717/peerj.6804 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Han et al. title: Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii link: https://peerj.com/articles/6834 last-modified: 2019-04-26 description: Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in Onchidium reevesii under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes, CYC, RPL28S, ACTB, TUBB, EF1a, Ubiq and 18S RNA, were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general, RPL28S, TUBB, ACTB and EF1a were ranked highly. ACTB and TUBB were found to be the most stable housekeeping genes under salinity stress, and EF1a plus TUBB was the most stable combination under injury stress. When analysing target gene expression in different tissues, RPL28S or EF1a should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions, ACTB (0 ppt, 5 ppt, 15 ppt, 25 ppt) and TUBB (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was TUBB plus EF1a. Therefore, we suggest that RPL28S, ACTB and TUBB are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of O. reevesii to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of O. reevesii under other stress conditions. creator: Teizhu Yang creator: Bingning Gu creator: Guolyu Xu creator: Yanmei Shi creator: Heding Shen creator: Rongcheng Rao creator: Hellen Lucas Mzuka uri: https://doi.org/10.7717/peerj.6834 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Yang et al. title: Melatonin improves the efficiency of super-ovulation and timed artificial insemination in sheep link: https://peerj.com/articles/6750 last-modified: 2019-04-25 description: It has been well proved that melatonin participates in the regulation of the seasonal reproduction of ewes. However, the effects of short term treatment of melatonin on ewe’s ovulation are still to be clarified. In this study, the effects of melatonin on the number of embryos harvested from superovulation, and the pregnant rate in recipients after embryo transferred have been investigated. Hu sheep with synchronous estrus treatment were given melatonin subcutaneously injection (0, 5, and 10 mg/ewe, respectively). It was found that the estrogen level in the group of 5 mg melatonin was significantly higher than that of other two groups at the time of sperm insemination (p < 0.05). The pregnant rate and number of lambs in the group of 5 mg melatonin treatment was also significantly higher than that of the rests of the groups (P < 0.05). In another study, 31 Suffolk ewes as donors and 103 small-tailed han sheep ewes as recipients were used to produce pronuclear embryo and embryo transfer. Melatonin (5 mg) was given to the donors during estrus. The results showed that, the number of pronuclear embryos and the pregnancy rate were also significantly higher in melatonin group than that in the control group. In addition, 28 donors and 44 recipient ewes were used to produce morula/blastocyst and embryo transferring. Melatonin (5 mg) was given during estrus. The total number of embryos harvested (7.40 ± 1.25/ewe vs. 3.96 ± 0.73/ewe, P < 0.05) and the pregnant rate (72.3 ± 4.6% vs. 54.7 ± 4.0%, P < 0.05) and number of lambs were also increased in melatonin group compared to the control group. Collectively, the results have suggested that melatonin treatment 36 hours after CIDR withdrawal could promote the number and quality of embryos in vivo condition and increased the pregnant rate and number of lambs. creator: Yukun Song creator: Hao Wu creator: Xuguang Wang creator: Aerman Haire creator: Xiaosheng Zhang creator: Jinlong Zhang creator: Yingjie Wu creator: Zhengxing Lian creator: Juncai Fu creator: Guoshi Liu creator: Abulizi Wusiman uri: https://doi.org/10.7717/peerj.6750 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Song et al. title: Management factors affecting physical health and welfare of tourist camp elephants in Thailand link: https://peerj.com/articles/6756 last-modified: 2019-04-25 description: BackgroundVariation in management across elephant camps likely has differential effects on the well-being of elephants.MethodsThis study calculated body condition, foot health and skin wound scores (WSs) for 122 elephants from 15 elephant camps in Chiang Mai province, and examined relationships to management factors using a multi-variable modeling approach.ResultsThe majority of elephants had high body condition scores (BCS) indicative of being overweight or obese, mild foot problems, but few visible wounds. Females had higher BCSs than males, as did elephants provided a water source at night. Increasing age was associated with higher foot and WSs. Higher WSs were observed in about a quarter of the cases where mahouts carried a hook. Wounds related to saddle riding were rare. Elephants that rested on sand floors at night had a decreased risk of high WSs compared to elephants that rested on compact dirt floors.DiscussionFindings emphasize the need for elephant camps to adjust management activities that negatively affect body condition (e.g., feeding too many sweet treats), foot health (e.g., hard substrates) and wounding (e.g., misuse of equipment) to improve health and welfare of this population. creator: Pakkanut Bansiddhi creator: Korakot Nganvongpanit creator: Janine L. Brown creator: Veerasak Punyapornwithaya creator: Pornsawan Pongsopawijit creator: Chatchote Thitaram uri: https://doi.org/10.7717/peerj.6756 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Bansiddhi et al. title: Diatom identification including life cycle stages through morphological and texture descriptors link: https://peerj.com/articles/6770 last-modified: 2019-04-25 description: Diatoms are unicellular algae present almost wherever there is water. Diatom identification has many applications in different fields of study, such as ecology, forensic science, etc. In environmental studies, algae can be used as a natural water quality indicator. The diatom life cycle consists of the set of stages that pass through the successive generations of each species from the initial to the senescent cells. Life cycle modeling is a complex process since in general the distribution of the parameter vectors that represent the variations that occur in this process is non-linear and of high dimensionality. In this paper, we propose to characterize the diatom life cycle by the main features that change during the algae life cycle, mainly the contour shape and the texture. Elliptical Fourier Descriptors (EFD) are used to describe the diatom contour while phase congruency and Gabor filters describe the inner ornamentation of the algae. The proposed method has been tested with a small algae dataset (eight different classes and more than 50 samples per type) using supervised and non-supervised classification techniques obtaining accuracy results up to 99% and 98% respectively. creator: Carlos Sánchez creator: Gabriel Cristóbal creator: Gloria Bueno uri: https://doi.org/10.7717/peerj.6770 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Sánchez et al. title: Biomass estimation equations for mesquite trees in the Americas link: https://peerj.com/articles/6782 last-modified: 2019-04-25 description: Mesquite trees are the preferred dendroenergy sources in arid and semi-arid forests. In spite of their relative importance, regional aboveground biomass (AGB) equations for mesquite trees are scarce in the scientific literature. For that reason, the aims of this study were to: (a) harvest trees and develop regional biomass equations; (b) contrast measured data with equations developed previously; and (c) test the applicability of the fitted equation for mesquite trees in the arid and semi-arid forests of the Americas. We harvested 206 new mesquite trees from arid and semi-arid forests in northern Mexico (Coahuila, Nuevo Leon, and Tamaulipas) in addition to using two other previously compiled data sets from Mexico (N = 304) to develop a regional equation. To test the validity of this equation, for biomass equations reported for the rest of the country, as well as for North and South American mesquite trees, we contrasted AGB measurements with predictions of fitted equations. Statistical analysis revealed the need for a single, regional, semi-empirical equation as together the three data sets represented the variability of the aboveground biomass of mesquite trees across northern Mexico, as well as mesquite trees in America’s arid and semiarid regions. Due to the large quantity of mesquite trees harvested for sampling and their variability, the regional biomass equation developed encompasses all other North and South American equations, and is representative of mesquite trees throughout the arid and semi-arid forests of the Americas. creator: Jose Navar creator: Felipa de Jesus Rodriguez-Flores creator: Julio Rios-Saucedo uri: https://doi.org/10.7717/peerj.6782 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Navar et al. title: Effects of complex training versus heavy resistance training on neuromuscular adaptation, running economy and 5-km performance in well-trained distance runners link: https://peerj.com/articles/6787 last-modified: 2019-04-25 description: BackgroundRecently, much attention has been paid to the role of neuromuscular function in long-distance running performance. Complex Training (CT) is a combination training method that alternates between performing heavy resistance exercises and plyometric exercises within one single session, resulting in great improvement in neuromuscular adaptation. The purpose of this study was to compare the effect of CT vs. heavy resistance training (HRT) on strength and power indicators, running economy (RE), and 5-km performance in well-trained male distance runners.MethodsTwenty-eight well-trained male distance runners (19–23 years old, VO2max:65.78 ± 4.99 ml.kg−1.min−1) performed one pre-test consisting of: maximum strength (1RM), counter movement jump (CMJ) height, peak power, a drop jump (DJ), and RE assessments, and blood lactate concentration (BLa) measurement at the speeds from 12–16 km.h−1, a 50-m sprint, and a 5-km running performance test. They were then divided into 3 groups: complex training group (CT, n = 10), that performed complex training and endurance training; heavy resistance training group (HRT, n = 9) that performed heavy strength training and endurance training; and control group (CON, n = 9) that performed strength-endurance training and endurance training. After the 8 weeks training intervention, all participants completed a post-test to investigate the training effects on the parameters measured.ResultsAfter training intervention, both the CT and HRT groups had improvements in: 1RM strength (16.88%, p < 0.001; 18.80%, p < 0.001, respectively), CMJ height (11.28%, p < 0.001; 8.96%, p < 0.001, respectively), 14 km.h−1RE (−7.68%, p < 0.001; −4.89%, p = 0.009, respectively), 50-m sprints (−2.26%, p = 0.003; −2.14%, p = 0.007, respectively) and 5-km running performance (−2.80%, p < 0.001; −2.09%, p < 0.001, respectively). The CON group did not show these improvements. All three training groups showed improvement in the 12 km.h−1RE (p ≤ 0.01). Only the CT group exhibited increases in DJ height (12.94%, p < 0.001), reactive strength index (19.99%, p < 0.001), 16 km.h−1 RE (−7.38%, p < 0.001), and a reduction of BLa concentrations at the speed of 16 km.h−1 (−40.80%, p < 0.001) between pre- and post-tests.ConclusionThis study demonstrated that CT can enhance 1RM strength, CMJ height, 12 and 14 km.h−1REs, 50-m sprints and 5-km running performances in well-trained male distance runners and may be superior to HRT for the development of reactive strength and 16 km.h−1RE, and reduction of BLa concentrations at speed of 16 km.h−1. Young male distance runners could integrate CT into their programs to improve the running performance. creator: Fei Li creator: Ran Wang creator: Robert U. Newton creator: David Sutton creator: Yue Shi creator: Haiyong Ding uri: https://doi.org/10.7717/peerj.6787 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Li et al. title: Long-read viral metagenomics captures abundant and microdiverse viral populations and their niche-defining genomic islands link: https://peerj.com/articles/6800 last-modified: 2019-04-25 description: Marine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in host culturing and a lack of reference genomes and ‘universal’ gene markers to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution, but their assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Such features also challenge assembly across genomic islands containing niche-defining genes that drive ecological speciation. These populations and features may be successfully captured by single-virus genomics and fosmid-based approaches, at least in abundant taxa, but at considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative sequencing and informatics workflow to improve viral metagenomic assemblies using short-read and long-read technology. The ‘VirION’ (Viral, long-read metagenomics via MinION sequencing) approach was first validated using mock communities where it was found to be as relatively quantitative as short-read methods and provided significant improvements in recovery of viral genomes. We then then applied VirION to the first metagenome from a natural viral community from the Western English Channel. In comparison to a short-read only approach, VirION: (i) increased number and completeness of assembled viral genomes; (ii) captured abundant, highly microdiverse virus populations, and (iii) captured more and longer genomic islands. Together, these findings suggest that VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature. creator: Joanna Warwick-Dugdale creator: Natalie Solonenko creator: Karen Moore creator: Lauren Chittick creator: Ann C. Gregory creator: Michael J. Allen creator: Matthew B. Sullivan creator: Ben Temperton uri: https://doi.org/10.7717/peerj.6800 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Warwick-Dugdale et al. title: Forensic characteristics and population genetics of Chinese Kazakh ethnic minority with an efficient STR panel link: https://peerj.com/articles/6802 last-modified: 2019-04-25 description: On the purpose of enhancing the forensic efficiency of CODIS STR loci, new STR loci have been gradually discovered and developed into some commercial multiplex systems. Recently, 22 STR loci including 18 non-CODIS STR loci and four CODIS STR loci were investigated in 501 unrelated healthy individuals of Kazakh ethnic group. Seven to 20 alleles at the different loci were identified and altogether 276 alleles for 22 selected loci were detected with the corresponding allelic frequencies ranging from 0.0010 to 0.3623. No significant deviation was observed from the Hardy–Weinberg equilibrium test for any of the 22 STRs. The value of cumulative power of discrimination in Kazakh group was 1-1.00E−28. Analyses of population differentiations and genetic distances between Kazakh and other Chinese groups presented that the Kazakh group with the Uygur group. These 22 STR loci evenly distributed on 22 different autosomal chromosomes were characterized by high genetic diversities and therefore could be utilized in the forensic cases to further increase the discrimination performance. creator: Chong Chen creator: Yuxin Guo creator: Xiaoye Jin creator: Wei Cui creator: Yuanyuan Wei creator: Yating Fang creator: Qiong Lan creator: Tingting Kong creator: Tong Xie creator: Bofeng Zhu uri: https://doi.org/10.7717/peerj.6802 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Chen et al. title: Beliefs are multidimensional and vary in stability over time - psychometric properties of the Beliefs and Values Inventory (BVI) link: https://peerj.com/articles/6819 last-modified: 2019-04-25 description: The cognitive processes underlying belief are still obscure. Understanding these processes may lead to more targeted treatment to better address functional impairment, such as occurs with delusions. One way in which this might be accomplished is to understand healthy, everyday beliefs, and how these may relate to characteristics observed in delusions. As yet, no such measure exists to accurately measure belief across a range of themes and dimensions. This paper outlines two studies documenting the creation and psychometric properties of a novel measure assessing three different dimensions of belief across themes of politics, science, the paranormal, religion, and morality in UK samples (n = 1, 673 total). Reliability estimates suggested good to excellent consistency (alpha > 0.8 per theme) with moderate to excellent reliability at 48 h (ICC = 0.61 –0.96) and 3.5 months (ICC = 0.61 –0.89). Factor analyses suggested good support for our five chosen themes of belief, suggesting they are distinct topic areas. Correlations across theme and dimension suggested dissociable characteristics within themes. These results have implications for 1. understanding the stability and relationship between themes of belief in a population and, 2. exploring how beliefs may change over time or as a result of an intervention. Full analysis code and data are available from the Open Science Framework (https://osf.io/hzvwr/). creator: Joseph M. Barnby creator: Vaughan Bell creator: Luke Sheridan Rains creator: Mitul A. Mehta creator: Quinton Deeley uri: https://doi.org/10.7717/peerj.6819 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Barnby et al. title: Hand hygiene knowledge and attitude of medical students in western Saudi Arabia link: https://peerj.com/articles/6823 last-modified: 2019-04-25 description: BackgroundThe practice of hand hygiene (HH) has prime importance among Health Care Professionals (HCPs) and non-compliance could cause adverse consequences. By keeping the importance of HH in mind, this study aims at investigating the knowledge and attitudes towards HH among medical students and interns at King Abdulaziz University (KAU), Jeddah, Saudi Arabia (SA).MethodsThe study includes 453 medical students and interns (189 males & 264 females). This study was completed in three months; from September to November 2017. The World Health Organization (WHO) HH questionnaire was used and data were analyzed on SPSS-21.ResultsTwo-third of the participants 292 (64.2%) had formal training in HH in the last three years. Almost half of the participants 254 (56.1%) had correct knowledge regarding the major course of transmission of potentially detrimental microbes among patients in the healthcare premises. Just 124 (27.4%) of the respondents had the correct knowledge about the most common basis of germs accountable for healthcare-related infections. Females had significantly better knowledge than males regarding the type of HH technique needed before palpation of the abdomen (177(54%) Vs. 151(46%); P < 0.002), before an injection (175(54.5%) Vs. 146(45.5%); P < 0.007), after emptying a bedpan (207(64.7%) Vs. 113(35.3%); P < 0.001), following discarding examination gloves (256(60.4% Vs. 168(39.6%); P < 0.001] and after exposure to blood (200(64.1%) Vs. 112(35.%); P < 0.001). Female participants had better knowledge than males regarding the type of HH actions. Females also had a significantly better attitude towards the importance of HH than males (240(62.5%) Vs. 144(37.5%); P < 0.001).ConclusionThe majority of the participants’ knowledge regarding HH was not up to the mark; however, female students had better knowledge compared to male students. The medical students and interns’ knowledge and positive attitude towards HH can play a pivotal role in preventing HCPs associated infections and it would overall decrease the infection-related burden on the hospital and governmental budgets. It is suggested that multi-dimensional plans are required to change this low compliance to a higher rate. creator: Marwan A. Bakarman creator: Mukhtiar Baig creator: Ahmad A. Malik creator: Zohair J. Gazzaz creator: Mostafa M. Mostafa creator: Mohamed A. Zayed creator: Abdulaziz S. Balubaid creator: Ahmed K. Alzahrani uri: https://doi.org/10.7717/peerj.6823 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Bakarman et al. title: Bibliometric analysis of scientific publications in rheumatology journals from China and other top-ranking countries between 2007 and 2017 link: https://peerj.com/articles/6825 last-modified: 2019-04-25 description: ObjectivesRheumatology-related diseases remain a significant burden worldwide. However, little is known about the comparative status of rheumatology research between Mainland China (MC) and the world’s leading countries. The aim of this study is to compare the quantity and quality of research output in the field of rheumatology that were written by researchers from MC, the USA, the UK, the Netherlands and France.MethodsBetween 2007 and 2017, all articles published in 30 rheumatology journals were identified via Science Citation Index Expanded database. The number of total and annual articles, article types (randomized controlled trials (RCTs), reviews, case reports, clinical trials and meta-analysis), impact factor (IF), citations, h-index and articles in the high-impact journals were collected for quantity and quality comparisons. The correlation of socioeconomic factors and annual publications was also analyzed.ResultsFrom 2007 to 2017, there were 53,439 articles published in rheumatology journals, of which researchers from the USA published 13,391 articles, followed by the UK, the Netherlands, France and MC with 6,179, 4,310, 4,066 and 2,898 articles, respectively. Publications from MC represented the ninth, but the number is growing rapidly. For total and average citations, MC still lags behind the other four countries in the study. Similar trends were observed in average IF, h-index and articles in the high-impact journals. In terms of article types, the USA occupies the dominant place, except for meta-analysis. The annual numbers of articles from MC and the USA were positively correlated with gross domestic product (p < 0.05).ConclusionsThe USA has played predominant role in rheumatology research for the last 11 years. The annual number of published articles from MC has increased notably from 2007 to 2017. Although MC has made progress in the number of published articles over the past decade, it still lags far behind the highly developed countries in most bibliometric indicators. Thus, the general quality of publications from MC needs further improvement. creator: Chen Zhang creator: Xinzhe Feng creator: Chen Wang creator: Denghui Liu creator: Chongru He creator: Weidong Xu uri: https://doi.org/10.7717/peerj.6825 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Zhang et al. title: Evolution of digestive enzymes and dietary diversification in birds link: https://peerj.com/articles/6840 last-modified: 2019-04-25 description: As the most species-rich class of tetrapod vertebrates, Aves possesses diverse feeding habits, with multiple origins of insectivory, carnivory, frugivory, nectarivory, granivory and omnivory. Since digestive enzymes mediate and limit energy and nutrient uptake, we hypothesized that genes encoding digestive enzymes have undergone adaptive evolution in birds. To test this general hypothesis, we identified 16 digestive enzyme genes (including seven carbohydrase genes (hepatic amy, pancreatic amy, salivary amy, agl, g6pc, gaa and gck), three lipase genes (cyp7a1, lipf and pnlip), two protease genes (ctrc and pgc), two lysozyme genes (lyz and lyg) and two chitinase genes (chia and chit1)) from the available genomes of 48 bird species. Among these 16 genes, three (salivary amy, lipf and chit1) were not found in all 48 avian genomes, which was further supported by our synteny analysis. Of the remaining 13 genes, eight were single-copy and five (chia, gaa, lyz, lyg and pgc) were multi-copy. Moreover, the multi-copy genes gaa, lyg and pgc were predicted to exhibit functional divergence among copies. Positively selected sites were detected in all of the analyzed digestive enzyme genes, except agl, g6pc, gaa and gck, suggesting that different diets may have favored differences in catalytic capacities of these enzymes. Furthermore, the analysis also revealed that the pancreatic amylase gene and one of the lipase genes (cyp7a1) have higher ω (the ratio of nonsynonymous to the synonymous substitution rates) values in species consuming a larger amount of seeds and meat, respectively, indicating an intense selection. In addition, the gck carbohydrase gene in species consuming a smaller amount of seeds, fruits or nectar, and a lipase gene (pnlip) in species consuming less meat were found to be under relaxed selection. Thus, gene loss, gene duplication, functional divergence, positive selection and relaxed selection have collectively shaped the evolution of digestive enzymes in birds, and the evolutionary flexibility of these enzymes may have facilitated their dietary diversification. creator: Yan-Hong Chen creator: Huabin Zhao uri: https://doi.org/10.7717/peerj.6840 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Chen and Zhao title: Training-induced increase in Achilles tendon stiffness affects tendon strain pattern during running link: https://peerj.com/articles/6764 last-modified: 2019-04-24 description: BackgroundDuring the stance phase of running, the elasticity of the Achilles tendon enables the utilisation of elastic energy and allows beneficial contractile conditions for the triceps surae muscles. However, the effect of changes in tendon mechanical properties induced by chronic loading is still poorly understood. We tested the hypothesis that a training-induced increase in Achilles tendon stiffness would result in reduced tendon strain during the stance phase of running, which would reduce fascicle strains in the triceps surae muscles, particularly in the mono-articular soleus.MethodsEleven subjects were assigned to a training group performing isometric single-leg plantarflexion contractions three times per week for ten weeks, and another ten subjects formed a control group. Before and after the training period, Achilles tendon stiffness was estimated, and muscle-tendon mechanics were assessed during running at preferred speed using ultrasonography, kinematics and kinetics.ResultsAchilles tendon stiffness increased by 18% (P < 0.01) in the training group, but the associated reduction in strain seen during isometric contractions was not statistically significant. Tendon elongation during the stance phase of running was similar after training, but tendon recoil was reduced by 30% (P < 0.01), while estimated tendon force remained unchanged. Neither gastrocnemius medialis nor soleus fascicle shortening during stance was affected by training.DiscussionThese results show that a training-induced increase in Achilles tendon stiffness altered tendon behaviour during running. Despite training-induced changes in tendon mechanical properties and recoil behaviour, the data suggest that fascicle shortening patterns were preserved for the running speed that we examined. The asymmetrical changes in tendon strain patterns supports the notion that simple in-series models do not fully explain the mechanical output of the muscle-tendon unit during a complex task like running. creator: Amelie Werkhausen creator: Neil J. Cronin creator: Kirsten Albracht creator: Gøran Paulsen creator: Askild V. Larsen creator: Jens Bojsen-Møller creator: Olivier R. Seynnes uri: https://doi.org/10.7717/peerj.6764 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Werkhausen et al. title: Likes, comments, and shares of marine organism imagery on Facebook link: https://peerj.com/articles/6795 last-modified: 2019-04-24 description: Several calls to action urge scientists and science communicators to engage more with online communities. While these calls have been answered by a high percentage of scientists and science communicators online, it often remains unclear what are the best models for effective communication. Best practices and methods for online science communication can benefit from experimental and quantitative research addressing how and when users engage with online content. This study addresses with quantitative and predictive models a key question for the popular, but often-ignored in science communication, social media platform Facebook. Specifically, this study examines the impact of imagery through quantification of likes, comments, and shares on Facebook posts. Here, I show that a basic quantitative model can be useful in predicting response to marine organism imagery on Facebook. The results of this online experiment suggest image type, novelty, and aesthetics impact the number of likes, shares, and comments on a post. In addition, the likes, shares, and comments on images did not follow traditional definitions of “charismatic megafauna”, with cephalopods and bony fishes receiving more interactions than cartilaginous fishes and marine mammals. Length and quality of caption did not significantly impact likes, comments, or shares. This study provides one of the first quantitative analysis of virality of scientific images via social media. The results challenge previously held conceptions of social media scientific outreach including increasing emphasis on imagery selection and curation, notions of which taxa the public connect with, and role of captions for imagery. creator: Craig R. McClain uri: https://doi.org/10.7717/peerj.6795 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 McClain title: Transcriptional analysis reveals the metabolic state of Burkholderia zhejiangensis CEIB S4-3 during methyl parathion degradation link: https://peerj.com/articles/6822 last-modified: 2019-04-24 description: Burkholderia zhejiangensis CEIB S4-3 has the ability to degrade methyl parathion (MP) and its main hydrolysis byproduct p-nitrophenol (PNP). According to genomic data, several genes related with metabolism of MP and PNP were identified in this strain. However, the metabolic state of the strain during the MP degradation has not been evaluated. In the present study, we analyzed gene expression changes during MP hydrolysis and PNP degradation through a transcriptomic approach. The transcriptional analysis revealed differential changes in the expression of genes involved in important cellular processes, such as energy production and conversion, transcription, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, among others. Transcriptomic data also exhibited the overexpression of both PNP-catabolic gene clusters (pnpABA′E1E2FDC and pnpE1E2FDC) present in the strain. We found and validated by quantitative reverse transcription polymerase chain reaction the expression of the methyl parathion degrading gene, as well as the genes responsible for PNP degradation contained in two clusters. This proves the MP degradation pathway by the strain tested in this work. The exposure to PNP activates, in the first instance, the expression of the transcriptional regulators multiple antibiotic resistance regulator and Isocitrate Lyase Regulator (IclR), which are important in the regulation of genes from aromatic compound catabolism, as well as the expression of genes that encode transporters, permeases, efflux pumps, and porins related to the resistance to multidrugs and other xenobiotics. In the presence of the pesticide, 997 differentially expressed genes grouped in 104 metabolic pathways were observed. This report is the first to describe the transcriptomic analysis of a strain of B. zhejiangensis during the biodegradation of PNP. creator: María Luisa Castrejón-Godínez creator: Ma. Laura Ortiz-Hernández creator: Emmanuel Salazar creator: Sergio Encarnación creator: Patricia Mussali-Galante creator: Efraín Tovar-Sánchez creator: Enrique Sánchez-Salinas creator: Alexis Rodríguez uri: https://doi.org/10.7717/peerj.6822 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Castrejón-Godínez et al. title: A fixed agreement—consequences of brood size manipulation on alternation in blue tits link: https://peerj.com/articles/6826 last-modified: 2019-04-24 description: Recent studies have proposed that conditional cooperation may resolve sexual conflict over the amount of care provided by each parent. Such conditional cooperation may allow parents to equalize their investment by alternating their provisioning visits. This alternated pattern of male and female visits, that is, alternation, is thought to stimulate each other’s investment leading to higher levels of provisioning and potential benefits for offspring development. However, experimental studies testing the role of alternation as an adaptive parental strategy to negotiate the level of investment are still absent. Therefore, we manipulated blue tit (Cyanistes caeruleus) parents by temporarily changing their brood sizes to induce changes in demand and thus visit rates. Parents were expected to visit more—assuming that prey sizes were constant—and alternate at higher levels when confronted with an enlarged brood given the greater potential for sexual conflict. In contrast, in reduced broods visit rates and alternation may become lower due to the smaller investment that is needed for reduced broods. We show that the level of alternation did not differ in response to the manipulated brood sizes, despite a directional change in visit rates for enlarged and reduced broods as expected. Nestlings did not benefit from high levels of alternation as no effects on nestling mass gain were present in either of the different manipulations. These findings indicate that alternation does not serve as a mechanism to motivate each other to feed at higher rates. Parents hence appeared to be inflexible in their level of alternation. We therefore suggest that the level of alternation might reflect a fixed agreement about the relative investment by each of the caring parents. creator: Maaike Griffioen creator: Wendt Müller creator: Arne Iserbyt uri: https://doi.org/10.7717/peerj.6826 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Griffioen et al. title: Normal spermatogenesis in Fank1 (fibronectin type 3 and ankyrin repeat domains 1) mutant mice link: https://peerj.com/articles/6827 last-modified: 2019-04-24 description: BackgroundThe fibronectin type 3 and ankyrin repeat domains 1 gene, Fank1, is an ancient, evolutionarily conserved gene present in vertebrates. Short-hairpin RNA (shRNA)-based knockdown transgenic mice have oligospermia caused by an increase in apoptotic germ cells. In this study, we investigated the in vivo function of Fank1.MethodsIn this study, we generated Fank1-knockout mice using the CRISPR/Cas9 system. We then investigated the phenotype and in vivo function of Fank1. Testes and epididymis tissues were analyzed by histological and immunofluorescence staining. Apoptotic cells were analyzed in terminal deoxynucleotidyl transferase dUTP nick end-labeling assays. Fertility and sperm counts were also evaluated. The GTEx database were used to assess gene expression quantitative trait loci and mRNA expression of candidate genes and genes neighboring single nucleotide polymorphisms was analyzed by quantitative RT-PCR.ResultsIn contrast to the Fank1-knockdown model, no significant changes in epididymal sperm content and the number of apoptotic cells were observed in Fank1−/− homozygotes. In addition, a different pattern of Dusp1, Klk1b21 and Klk1b27 mRNA expression was detected in Fank1-knockout testis. These results reveal differences in the molecular changes between Fank1-knockdown mice and Fank1-knockout mice and provide a basic resource for population genetics studies. creator: Jintao Zhang creator: Xin Zhang creator: Yue Zhang creator: Wentao Zeng creator: Shuqin Zhao creator: Mingxi Liu uri: https://doi.org/10.7717/peerj.6827 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Zhang et al. title: Periodontal condition in growing subjects with Marfan Syndrome: a case-control study link: https://peerj.com/articles/6606 last-modified: 2019-04-23 description: BackgroundMarfan’s syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in the extracellular matrix protein fibrillin-1. Orofacial characteristics may be useful in identification of the syndrome. Severe periodontitis is sometimes observed in MFS patients, but no in-depth information has been reported in Italian groups of growing subjects with MFS. The aim of this study was to analyze the periodontal condition on a group of growing subjects affected by MFS, in comparison with a typically developed control group.MethodsA group of 16 subjects with diagnosed MFS were recruited from the Centre for Rare Diseases for Marfan Syndrome and Related Disorders of Tor Vergata University Hospital. The Marfan Group (MG) was compared with a Control Group (CG) composed by 20 nonsyndromic subjects. The periodontal clinical parameters like Marginal Gingival Thickness (GT), Plaque Index (PI), Bleeding On Probing (BOP) and Modified Periodontal Screening and Recording (PSR) were assessed.ResultsThe mean value of PI in MG was 59%, instead in CG it reached 21%. Analysis showed a significant difference between MG and CG also for the BOP. In MG the mean value of BOP attained 36% and in CG it reached 16%. A statistical significant difference of distribution of PSR index between the two groups was found for all sextant examined.DiscussionPatients with Marfan syndrome reveal a higher presence of plaque and consequently a generalized inflammation in the oral cavity when compared with a control group. creator: Nicolò Venza creator: Carlotta Danesi creator: Diego Contò creator: Francesco Fabi creator: Gianluca Mampieri creator: Federica Sangiuolo creator: Giuseppina Laganà uri: https://doi.org/10.7717/peerj.6606 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Venza et al. title: Preference classes in society for coastal marine protected areas link: https://peerj.com/articles/6672 last-modified: 2019-04-23 description: Marine protected areas (MPAs) are increasingly being used as conservation tools in the marine environment. Success of MPAs depends upon sound scientific design and societal support. Studies that have assessed societal preferences for temperate MPAs have generally done it without considering the existence of discrete groups of opinion within society and have largely considered offshore and deep-sea areas. This study quantifies societal preferences and economic support for coastal MPAs in Wales (UK) and assesses the presence of distinct groups of preference for MPA management, through a latent class choice experiment approach. Results show a general support for the protection of the marine environment in the form of MPAs and that society is willing to bear the costs derived from conservation. Despite a general opposition toward MPAs where human activities are completely excluded, there is some indication that three classes of preferences within society can be established regarding the management of potentially sea-floor damaging activities. This type of approach allows for the distinction between those respondents with positive preferences for particular types of management from those who experience disutility. We conclude that insights from these types of analyses can be used by policy-makers to identify those MPA designs and management combinations most likely to be supported by particular sectors of society. creator: Ana Ruiz-Frau creator: James M. Gibbons creator: Hilmar Hinz creator: Gareth Edwards-Jones creator: Michel J. Kaiser uri: https://doi.org/10.7717/peerj.6672 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Ruiz-Frau et al. title: Introgressive hybridization in a Spiny-Tailed Iguana, Ctenosaura pectinata, and its implications for taxonomy and conservation link: https://peerj.com/articles/6744 last-modified: 2019-04-23 description: Introgression, the transmission of genetic material of one taxon into another through hybridization, can have various evolutionary outcomes. Previous studies have detected signs of introgression between western populations of the Mexican endemic and threatened spiny-tailed iguana, Ctenosaura pectinata. However, the extent of this phenomenon along the geographic distribution of the species is unknown. Here, we use multilocus data together with detailed geographic sampling to (1) define genotypic clusters within C. pectinata; (2) evaluate geographic concordance between maternally and biparentally inherited markers; (3) examine levels of introgression between genotypic clusters, and (4) suggest taxonomic modifications in light of this information. Applying clustering methods to genotypes of 341 individuals from 49 localities of C. pectinata and the closely related C. acanthura, we inferred the existence of five genotypic clusters. Contact zones between genotypic clusters with signatures of interbreeding were detected, showing different levels of geographic discordance with mtDNA lineages. In northern localities, mtDNA and microsatellites exhibit concordant distributions, supporting the resurrection of C. brachylopha. Similar concordance is observed along the distribution of C. acanthura, confirming its unique taxonomic identity. Genetic and geographic concordance is also observed for populations within southwestern Mexico, where the recognition of a new species awaits in depth taxonomic revision. In contrast, in western localities a striking pattern of discordance was detected where up to six mtDNA lineages co-occur with only two genotypic clusters. Given that the type specimen originated from this area, we suggest that individuals from western Mexico keep the name C. pectinata. Our results have profound implications for conservation, management, and forensics of Mexican iguanas. creator: Eugenia Zarza creator: Víctor H. Reynoso creator: Christiana M. A. Faria creator: Brent C. Emerson uri: https://doi.org/10.7717/peerj.6744 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Zarza et al. title: Noblella thiuni sp. n., a new (singleton) species of minute terrestrial-breeding frog (Amphibia, Anura, Strabomantidae) from the montane forest of the Amazonian Andes of Puno, Peru link: https://peerj.com/articles/6780 last-modified: 2019-04-23 description: We describe a new species of minute, terrestrial-breeding frog in the genus Noblella. We collected a single specimen in the leaf litter of primary montane forest (2,225 m a.s.l.) near Thiuni, in the Provice of Carabaya, Department of Puno, in the upper watershed of a tributary of the Inambari River of southern Peru, the same locality where we found the types of Psychrophrynella glauca Catenazzi & Ttito 2018. We placed the new species within Noblella on the basis of molecular data, minute size, and overall morphological resemblance with the type species N. peruviana and other species of Noblella, including having three phalanges on finger IV (as in N. coloma, N. heyeri, N. lynchi, N. madreselva, N. peruviana, and N. pygmaea), and terminal phalanges T-shaped and pointed. Noblella thiuni sp. n. is distinguished from all other species of Noblella by having ventral surfaces of legs bright red, and chest and belly copper reddish with a profusion of silvery spots. The new species further differs from known Peruvian species of Noblella by the combination of the following characters: tympanic membrane absent, eyelids lacking tubercles, dorsal skin finely shagreen, tarsal tubercles or folds absent, three phalanges on Finger IV, tips of digits not expanded, no circumferential grooves on digits, inguinal spots present. The new species has a snout–vent length of 11.0 mm in one adult or subadult male. Our new finding confirms the high levels of endemism and beta diversity of small, terrestrial-breeding frogs inhabiting the moss layers and leaf litter in the montane forests of the Amazonian slopes of the Andes and adjacent moist puna grasslands, and suggests much work remains to be done to properly document this diversity. creator: Alessandro Catenazzi creator: Alex Ttito uri: https://doi.org/10.7717/peerj.6780 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Catenazzi and Ttito title: A network approach to identify bioregions in the distribution of Mediterranean amphipods associated with Posidonia oceanica meadows link: https://peerj.com/articles/6786 last-modified: 2019-04-23 description: Although amphipods are key components of the macro-fauna associated with Posidonia oceanica meadows, to date no studies focused on the structure and diversity of their assemblages across the whole Mediterranean Sea. Here, we applied a network approach based on modularity on a dataset mined from literature to identify biogeographic modules and to assess the biogeographic roles of associated localities. We also correlated the patterns evidenced with the biogeographic distribution of amphipod groups by means of a multivariate analysis. Modularity analysis highlighted four biogeographic modules bounded by the main Mediterranean biogeographic divides and evidenced a decrease in species diversity along a NW-SE gradient. Assemblages associated with Central-Western Mediterranean and, to a lesser extent, Tunisian modules showed the highest species richness and were identified as hubs, characterized by species with regional distributions that behave as source in a biogeographic context. The paleogeographic history of the host seagrass and the ecology of associated amphipods, both suggest the joint effect of species persistence and post-Last Glacial Maximum expansion in explaining the pattern of amphipod distribution in the Mediterranean Sea. creator: Bruno Bellisario creator: Federica Camisa creator: Chiara Abbattista creator: Roberta Cimmaruta uri: https://doi.org/10.7717/peerj.6786 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Bellisario et al. title: Gastrocnemius fascicles are shorter and more pennate throughout the first month following acute Achilles tendon rupture link: https://peerj.com/articles/6788 last-modified: 2019-04-23 description: The purpose of this study was to characterize the short-term effects of Achilles tendon ruptures on medial gastrocnemius. We hypothesized that the fascicles of the medial gastrocnemius muscle of the injured Achilles tendon would be shorter and more pennate immediately following the injury and would persist throughout 4 weeks post-injury. B-mode longitudinal ultrasound images of the medial gastrocnemius were acquired in 10 adults who suffered acute Achilles tendon ruptures and were treated non-operatively. Ultrasound images were acquired during the initial clinical visit following injury as well as 2 and 4 weeks following this initial clinical visit. Resting muscle structure was characterized by measuring fascicle length, pennation angle, muscle thickness, and muscle echo intensity in both the injured and contralateral (control) limbs. Fascicle length was 15% shorter (P < 0.001) and pennation angle was 21% greater (P < 0.001) in the injured muscle compared to the uninjured (control) muscle at the presentation of injury (week 0). These differences in fascicle length persisted through 4 weeks after injury (P < 0.002) and pennation angle returned to pre-injury levels. Muscle thickness changes were not detected at any of the post-injury visits (difference < 4%, P > 0.026). Echo intensity of the injured limb was 8% lower at the presentation of the injury but was not different compared to the contralateral muscle at 2 and 4 weeks post-injury. Our results suggest that Achilles tendon ruptures elicit rapid changes in the configuration of the medial gastrocnemius, which may explain long-term functional deficits. creator: Todd J. Hullfish creator: Kathryn M. O’Connor creator: Josh R. Baxter uri: https://doi.org/10.7717/peerj.6788 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Hullfish et al. title: The effect of pharmacological inhibition of Serine Proteases on neuronal networks in vitro link: https://peerj.com/articles/6796 last-modified: 2019-04-23 description: Neurons are embedded in an extracellular matrix (ECM), which functions both as a scaffold and as a regulator of neuronal function. The ECM is in turn dynamically altered through the action of serine proteases, which break down its constituents. This pathway has been implicated in the regulation of synaptic plasticity and of neuronal intrinsic excitability. In this study, we determined the short-term effects of interfering with proteolytic processes in the ECM, with a newly developed serine protease inhibitor. We monitored the spontaneous electrophysiological activity of in vitro primary rat cortical cultures, using microelectrode arrays. While pharmacological inhibition at a low dosage had no significant effect, at elevated concentrations it altered significantly network synchronization and functional connectivity but left unaltered single-cell electrical properties. These results suggest that serine protease inhibition affects synaptic properties, likely through its actions on the ECM. creator: Sebastiaan Van De Vijver creator: Stephan Missault creator: Jeroen Van Soom creator: Pieter Van Der Veken creator: Koen Augustyns creator: Jurgen Joossens creator: Stefanie Dedeurwaerdere creator: Michele Giugliano uri: https://doi.org/10.7717/peerj.6796 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Van De Vijver et al. title: CYP2C11 played a significant role in down-regulating rat blood pressure under the challenge of a high-salt diet link: https://peerj.com/articles/6807 last-modified: 2019-04-23 description: BackgroundArachidonic acid (AA) is oxidized by cytochrome P450s (CYPs) to form epoxyeicosatrienoic acids (EETs), compounds that modulate ion transport, gene expression, and vasorelaxation. Both CYP2Cs and CYP2Js are involved in kidney EET epoxidation.MethodsIn this study, we used a CYP2C11-null rat model to explore the in vivo effects of CYP2C11 on vasorelaxation. For 2 months, CYP2C11-null and wild-type (WT) Sprague-Dawley rats were either fed normal lab (0.3% (w/w) sodium chloride) or high-salt (8% (w/w) sodium chloride) diets. Subsequently, an invasive method was used to determine blood pressure. Next, western blots, quantitative PCR, and immunohistochemistry were used to determine renal expression of CYPs involved in AA metabolism.ResultsAmong CYP2C11-null rats, a high-salt diet (females: 156.79 ± 15.89 mm Hg, males: 130.25 ± 16.76 mm Hg, n = 10) resulted in significantly higher blood pressure than a normal diet (females: 118.05 ± 8.43 mm Hg, P < 0.01; males: 115.15 ± 11.45 mm Hg, P < 0.05, n = 10). Compared with WT rats under the high-salt diet, western blots showed that CYP2C11-null rats had higher renal expression of CYP2J2 and CYP4A. This was consistent with the results of immunohistochemistry and the qPCR, respectively. The two rat strains did not differ in the renal expression of CYP2C23 or CYP2C24.ConclusionOur findings suggested that CYP2C11 plays an important role in lowering blood pressure under the challenge of a high-salt diet. creator: Wei Liu creator: Danjuan Sui creator: Huanying Ye creator: Zhen Ouyang creator: Yuan Wei uri: https://doi.org/10.7717/peerj.6807 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Liu et al. title: Identification of a putative competitive endogenous RNA network for lung adenocarcinoma using TCGA datasets link: https://peerj.com/articles/6809 last-modified: 2019-04-23 description: The mechanisms underlying the oncogenesis and progression of lung adenocarcinoma (LUAD) are currently unclear. The discovery of competitive endogenous RNA (ceRNA) regulatory networks has provided a new direction for the treatment and prognosis of patients with LUAD. However, the mechanism of action of ceRNA in LUAD remains elusive. In the present study, differentially expressed mRNAs, microRNAs (miRs) and long non-coding RNAs from the cancer genome atlas database were screened. CeRNAs for LUAD were then identified using online prediction software. Among the ceRNAs identified, family with sequence similarity 83 member A (FAM83A), miR-34c-5p, KCNQ1OT1 and FLJ26245 were observed to be significantly associated with the overall survival of patients with LUAD. Of note, FAM83A has potential significance in drug resistance, and may present a candidate biomarker for the prognosis and treatment of patients with LUAD. creator: Yuanyong Wang creator: Tong Lu creator: Yang Wo creator: Xiao Sun creator: Shicheng Li creator: Shuncheng Miao creator: Yanting Dong creator: Xiaoliang Leng creator: Wenjie Jiao uri: https://doi.org/10.7717/peerj.6809 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Wang et al. title: Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations link: https://peerj.com/articles/6829 last-modified: 2019-04-23 description: BackgroundQuantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for quantifying mRNA abundance. With relative expression analysis, however, reliable data output is dependent on stably expressed reference genes across the samples being studied. In anorectal malformations (ARMs), there is limited data on the selection of appropriate reference genes.PurposeThis study was aimed to investigate the optimal reference genes for PCR in ARM rat models.MethodsWe selected 15 commonly used reference genes (Rps18, Actb, B2m, Gapdh, Ppia, Hprt1, Pgk1, Ywhaz, Tbp, Ubc, Rps16, Rpl13a, Rplp1, Sdha, and Hmbs) as candidate reference genes and detected their mRNA expression in ARM samples by RT-qPCR. The expression stability and variability of these transcripts were subsequently evaluated using four methods (geNorm, NormFinder, comparative ΔCt, and BestKeeper).ResultsThe abundance of the candidate reference genes was qualified by RT-qPCR and the cycle threshold (Ct) values ranged between 14.07 (Rplp1) and 21.89 (Sdha). In the overall candidate genes, different variations existed across the different algorithms. A comprehensive analysis revealed that Rpl13a ranked first among the relatively stable genes, followed by Ywhaz, Rps18, Sdha, and Hmbs.ConclusionsThe most stable reference genes for RT-qPCR were Rpl13a, Ywhaz, and Rps18 in ETU-induced ARMs in rat fetus. This study provided a foundation for reference gene selection for future gene expression analyses. creator: Caiyun Long creator: Yunxia Xiao creator: Siying Li creator: Xiaobing Tang creator: Zhengwei Yuan creator: Yuzuo Bai uri: https://doi.org/10.7717/peerj.6829 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Long et al. title: Next-generation sequencing of BRCA1 and BRCA2 genes for rapid detection of germline mutations in hereditary breast/ovarian cancer link: https://peerj.com/articles/6661 last-modified: 2019-04-22 description: BackgroundConventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment.MethodsWe first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set).ResultsBy minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis.DiscussionOur experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches. creator: Arianna Nicolussi creator: Francesca Belardinilli creator: Yasaman Mahdavian creator: Valeria Colicchia creator: Sonia D’Inzeo creator: Marialaura Petroni creator: Massimo Zani creator: Sergio Ferraro creator: Virginia Valentini creator: Laura Ottini creator: Giuseppe Giannini creator: Carlo Capalbo creator: Anna Coppa uri: https://doi.org/10.7717/peerj.6661 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Nicolussi et al. title: MicroRNA profiling of mouse liver in response to DENV-1 infection by deep sequencing link: https://peerj.com/articles/6697 last-modified: 2019-04-22 description: BackgroundDengue caused by dengue virus (DENV) serotypes −1 to −4 is the most important mosquito-borne viral disease in the tropical and sub-tropical countries worldwide. Yet many of the pathophysiological mechanisms of host responses during DENV infection remain largely unknown and incompletely understood.MethodsUsing a mouse model, the miRNA expressions in liver during DENV-1 infection was investigated using high throughput miRNA sequencing. The differential expressions of miRNAs were then validated by qPCR, followed by target genes prediction. The identified miRNA targets were subjected to gene ontology (GO) annotation and pathway enrichment analysis to elucidate the potential biological pathways and molecular mechanisms associated with DENV-1 infection.ResultsA total of 224 and 372 miRNAs out of 433 known mouse miRNAs were detected in the livers of DENV-1-infected and uninfected mice, respectively; of these, 207 miRNAs were present in both libraries. The miR-148a-3p and miR-122-5p were the two most abundant miRNAs in both groups. Thirty-one miRNAs were found to have at least 2-fold change in upregulation or downregulation, in which seven miRNAs were upregulated and 24 miRNAs were downregulated in the DENV-1-infected mouse livers. The miR-1a-3p was found to be the most downregulated miRNA in the DENV-1-infected mouse livers, with a significant fold change of 0.10. To validate the miRNA sequencing result, the expression pattern of 12 miRNAs, which were highly differentially expressed or most abundant, were assessed by qPCR and nine of them correlated positively with the one observed in deep sequencing. In silico functional analysis revealed that the adaptive immune responses involving TGF-beta, MAPK, PI3K-Akt, Rap1, Wnt and Ras signalling pathways were modulated collectively by 23 highly differentially expressed miRNAs during DENV-1 infection.ConclusionThis study provides the first insight into the global miRNA expressions of mouse livers in response to DENV-1 infection in vivo and the possible roles of miRNAs in modulating the adaptive immune responses during DENV-1 infection. creator: Lian Yih Pong creator: Sinikka Parkkinen creator: Amreeta Dhanoa creator: Han Ming Gan creator: Indeevari Abisheka Chiharu Wickremesinghe creator: Sharifah Syed Hassan uri: https://doi.org/10.7717/peerj.6697 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Pong et al. title: Integrated analysis of two-lncRNA signature as a potential prognostic biomarker in cervical cancer: a study based on public database link: https://peerj.com/articles/6761 last-modified: 2019-04-22 description: BackgroundCervical cancer (CC) is a common gynecological malignancy in women worldwide. Evidence suggests that long non-coding RNAs (lncRNAs) can be used as biomarkers in patients with CC. However, prognostic biomarkers for CC are still lacking. The aim of our study was to find lncRNA biomarkers which are able to predict prognosis in CC based on the data from The Cancer Genome Atlas (TCGA).MethodsThe patients were divided into three groups according to FIGO stage. Differentially expressed lncRNAs were identified in CC tissue compared to adjacent normal tissues based on a fold change >2 and <0.5 at P < 0.05 for up- and downregulated lncRNA, respectively. The relationship between survival outcome and lncRNA expression was assessed with univariate and multivariate Cox proportional hazards regression analysis. We constructed a risk score as a method to evaluate prognosis. We used receiver operating characteristic (ROC) curve and the area under curve (AUC) analyses to assess the diagnostic value of a two-lncRNA signature. We detected the expression levels of the two lncRNAs in 31 pairs of newly diagnosed CC specimens and paired adjacent non-cancerous tissue specimens, and also in CC cell lines. Finally, the results were statistically compared using t-tests.ResultsIn total, 289 RNA sequencing profiles and accompanying clinical data were obtained. We identified 49 differentially expressed lncRNAs, of which two related to overall survival (OS) in CC patients. These two lncRNAs (ILF3-AS1 and RASA4CP) were found together as a single prognostic signature. Meanwhile, the prognosis of patients with low-risk CC was better and positively correlated with OS (P < 0.001). Further analysis showed that the combined two-lncRNA expression signature could be used as an independent biomarker to evaluate the prognosis in CC. qRT-PCR results were consistent with TCGA, confirming downregulated expression of both lncRNAs. Furthermore, upon ROC curve analysis, the AUC of the combined lncRNAs was greater than that of the single lncRNAs alone (0.723 vs 0.704 and 0.685), respectively; P < 0.05.ConclusionsOur study showed that the two-lncRNA signature of ILF3-AS1 and RASA4CP can be used as an independent biomarker for the prognosis of CC, based on bioinformatic analysis. creator: Wenjuan Wu creator: Jing Sui creator: Tong Liu creator: Sheng Yang creator: Siyi Xu creator: Man Zhang creator: Shaoping Huang creator: Lihong Yin creator: Yuepu Pu creator: Geyu Liang uri: https://doi.org/10.7717/peerj.6761 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Wu et al. title: Extinction and the U.S. Endangered Species Act link: https://peerj.com/articles/6803 last-modified: 2019-04-22 description: The U.S. Endangered Species Act is one of the strongest laws of any nation for preventing species extinction, but quantifying the Act’s effectiveness has proven difficult. To provide one measure of effectiveness, we identified listed species that have gone extinct and used previously developed methods to update an estimate of the number of species extinctions prevented by the Act. To date, only four species have been confirmed extinct with another 22 possibly extinct following protection. Another 71 listed species are extinct or possibly extinct, but were last seen before protections were enacted, meaning the Act’s protections never had the opportunity to save these species. In contrast, a total of 39 species have been fully recovered, including 23 in the last 10 years. We estimate the Endangered Species Act has prevented the extinction of roughly 291 species since passage in 1973, and has to date saved more than 99% of species under its protection. creator: Noah Greenwald creator: Kieran F. Suckling creator: Brett Hartl creator: Loyal A. Mehrhoff uri: https://doi.org/10.7717/peerj.6803 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Greenwald et al. title: eHealth adoption and use among healthcare professionals in a tertiary hospital in Sub-Saharan Africa: a Qmethodology study link: https://peerj.com/articles/6326 last-modified: 2019-04-19 description: BackgroundThe aim of the study was to explore the viewpoints of healthcare professionals (HCPs) on the adoption and use of eHealth in clinical practice in sub-Saharan Africa (SSA). Information and communication technologies (ICTs) including eHealth provide HCPs the opportunity to provide quality healthcare to their patients while also improving their own clinical practices. Despite this, previous research has identified these technologies have their associated challenges when adopting them for clinical practice. But more research is needed to identify how these eHealth resources influence clinical practice. In addition, there is still little information about adoption and use of these technologies by HCPs inclinical practice in Sub-Saharan Africa.MethodAn exploratory descriptive design was adopted for this study. Thirty-six (36) HCPs (18 nurses and 18 physicians) working in the clinical area in a tertiary health institution in SSA participated in this study. Using Qmethodology, study participants rank-ordered forty-six statementsin relation to their adoption and use of eHealth within their clinical practice.This was analysed using by-person factor analysis and complemented with audio-taped interviews.ResultsThe analysis yielded four factors i.e., distinct viewpoints the HCPs hold about adoption and use of eHealth within their clinical practice. These factors include: “Patient-focused eHealth advocates” who use the eHealth because they are motivated by patients and their families preferences; “Task-focused eHealth advocates” use eHealth because it helps them complete clinical tasks; “Traditionalistic-pragmatists” recognise contributions eHealth makes in clinical practice but separate from their routine clinical activities; and the “Tech-focused eHealth advocates” who use the eHealth because they are motivated by the technology itself.ConclusionThe study shows the equivocal viewpoints that HCPs have about eHealth within their clinical practice. This, in addition to adding to existing literature, will help policymakers/decision makers to consider HCPs views about these technologies prior to implementing an eHealth resource. creator: Muhammad Awwal Ladan creator: Heather Wharrad creator: Richard Windle uri: https://doi.org/10.7717/peerj.6326 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Ladan et al. title: Rapid ecological specialization despite constant population sizes link: https://peerj.com/articles/6476 last-modified: 2019-04-19 description: BackgroundThe bushbuck, Tragelaphus scriptus, is a widespread and ecologically diverse ungulate species complex within the spiral-horned antelopes. This species was recently found to consist of two genetically divergent but monophyletic lineages, which are paraphyletic at mitochondrial (mt)DNA owing to an ancient interspecific hybridization event. The Scriptus lineage (T. s. scriptus) inhabits the north-western half of the African continent while Sylvaticus (T. s. sylvaticus) is found in the south-eastern half. Here we test hypotheses of historical demography and adaptation in bushbuck using a higher-resolution framework, with four nuclear (MGF, PRKCI, SPTBN, and THY) and three new mitochondrial markers (cytochrome b, 12S rRNA, and 16S rRNA).MethodsGenealogies were reconstructed for the mitochondrial and nuclear data sets, with the latter dated using fossil calibration points. We also inferred the demographic history of Scriptus and Sylvaticus using coalescent-based methods. To obtain an overview of the origins and ancestral colonisation routes of ancestral bushbuck sequences across geographic space, we conducted discrete Bayesian phylogeographic and statistical dispersal-vicariance analyses on our nuclear DNA data set.ResultsBoth nuclear DNA and mtDNA support previous findings of two genetically divergent Sylvaticus and Scriptus lineages. The three mtDNA loci confirmed 15 of the previously defined haplogroups, including those with convergent phenotypes. However, the nuclear tree showed less phylogenetic resolution at the more derived parts of the genealogy, possibly due to incomplete lineage sorting of the slower evolving nuclear genome. The only exception to this was the montane Menelik’s bushbuck (Sylvaticus) of the Ethiopian highlands, which formed a monophyletic group at three of four nuclear DNA loci. We dated the coalescence of the two lineages to a common ancestor ∼2.54 million years ago. Both marker sets revealed similar demographic histories of constant population size over time. We show that the bushbuck likely originated in East Africa, with Scriptus dispersing to colonise suitable habitats west of the African Rift and Sylvaticus radiating from east of the Rift into southern Africa via a series of mainly vicariance events.DiscussionDespite lower levels of genetic structure at nuclear loci, we confirmed the independent evolution of the Menelik’s bushbuck relative to the phenotypically similar montane bushbuck in East Africa, adding further weight to previous suggestions of convergent evolution within the bushbuck complex. Perhaps the most surprising result of our analysis was that both Scriptus and Sylvaticus populations remained relatively constant throughout the Pleistocene, which is remarkable given that this was a period of major climatic and tectonic change in Africa, and responsible for driving the evolution of much of the continent’s extant large mammalian diversity. creator: Andrinajoro R. Rakotoarivelo creator: Paul O’Donoghue creator: Michael W. Bruford creator: Yoshan Moodley uri: https://doi.org/10.7717/peerj.6476 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Rakotoarivelo et al. title: Ability emotional intelligence in young people and older adults with and without depressive symptoms, considering gender and educational level link: https://peerj.com/articles/6595 last-modified: 2019-04-19 description: BackgroundThere is little research on differences in Emotional Intelligence (EI) ability at different stages of adult development. The few published studies tend not to use older adult samples. Previous studies on EI ability and age have shown contradictory results. Our main objective was to evaluate results in EI ability across different stages of adult development, taking into account gender, depressive symptoms, and educational level.MethodsWe interviewed 166 participants (108 women), 66 of whom were aged 18–30 years, 53 aged 31–60 years, and 40 aged 61–76 years. All were either working or enrolled in colleges at the time of the study. The assessment tools used were the Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT), a test that assesses performance-based EI, and the Centre for Epidemiologic Studies-Depression (CES-D) scale, a tool to evaluate depressive symptoms.ResultsYoung people, women, and participants with a higher educational level achieved higher scores on the MSCEIT. Additionally, depressive symptomatology was only partially associated with the MSCEIT (i.e., with the using emotions branch). However, a subsequent joint analysis of the independent effects of variables age, gender, educational level, and depressive symptomatology and their interactions on MSCEIT total suggests that only educational level and depressive symptomatology were associated with EI ability, with the direct relationship between age and gender with MSCEIT disappearing. Additionally, our study indicated an interaction effect between age and depressive symptoms, showing that participants in age cohorts 18–30 and 31–60 and without depressive symptoms have a higher EI ability.DiscussionOur study suggests that the direct effects of age and gender on EI ability across adult development, using a wide age range, can change or disappear when effects of educational level and depressive symptomatology, and their interactions, are controlled for. Our results also suggest that EI ability is a protective factor against depression in some age cohorts. This novel aspect of our study does not appear in the previous literature. However, prospective studies are needed to verify these findings and examine whether other psychological variables could determine the relations between age, gender and EI ability across adult development. creator: Beatriz Navarro-Bravo creator: José M. Latorre creator: Ana Jiménez creator: Rosario Cabello creator: Pablo Fernández-Berrocal uri: https://doi.org/10.7717/peerj.6595 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Navarro-Bravo et al. title: The newly described Araguaian river dolphins, Inia araguaiaensis (Cetartiodactyla, Iniidae), produce a diverse repertoire of acoustic signals link: https://peerj.com/articles/6670 last-modified: 2019-04-19 description: The recent discovery of the Araguaian river dolphin (Inia araguaiaensis) highlights how little we know about the diversity and biology of river dolphins. In this study, we described the acoustic repertoire of this newly discovered species in concert with their behaviour. We analysed frequency contours of 727 signals (sampled at 10 ms temporal resolution). These contours were analyzed using an adaptive resonance theory neural network combined with dynamic time-warping (ARTwarp). Using a critical similarity value of 96%, frequency contours were categorized into 237 sound-types. The most common types were emitted when calves were present suggesting a key role in mother-calf communication. Our findings show that the acoustic repertoire of river dolphins is far from simple. Furthermore, the calls described here are similar in acoustic structure to those produced by social delphinids, such as orcas and pilot whales. Uncovering the context in which these signals are produced may help understand the social structure of this species and contribute to our understanding of the evolution of acoustic communication in whales. creator: Gabriel Melo-Santos creator: Angélica Lúcia Figueiredo Rodrigues creator: Rodrigo Hipólito Tardin creator: Israel de Sá Maciel creator: Miriam Marmontel creator: Maria Luisa Da Silva creator: Laura Johanna May-Collado uri: https://doi.org/10.7717/peerj.6670 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Melo-Santos et al. title: Physiological responses of Oxyrrhis marina to a diet of virally infected Emiliania huxleyi link: https://peerj.com/articles/6722 last-modified: 2019-04-19 description: The coccolithophore Emiliania huxleyi forms some of the largest phytoplankton blooms in the ocean. The rapid demise of these blooms has been linked to viral infections. E. huxleyi abundance, distribution, and nutritional status make them an important food source for the heterotrophic protists which are classified as microzooplankton in marine food webs. In this study we investigated the fate of E. huxleyi (CCMP 374) infected with virus strain EhV-86 in a simple predator-prey interaction. The ingestion rates of Oxyrrhis marina were significantly lower (between 26.9 and 50.4%) when fed virus-infected E. huxleyi cells compared to non-infected cells. Despite the lower ingestion rates, O. marina showed significantly higher growth rates (between 30 and 91.3%) when fed infected E. huxleyi cells, suggesting higher nutritional value and/or greater assimilation of infected E. huxleyi cells. No significant differences were found in O. marina cell volumes or fatty acids profiles. These results show that virally infected E. huxleyi support higher growth rates of single celled heterotrophs and in addition to the “viral shunt” hypothesis, viral infections may also divert more carbon to mesozooplankton grazers. creator: Andrew G. Goode creator: David M. Fields creator: Stephen D. Archer creator: Joaquín Martínez Martínez uri: https://doi.org/10.7717/peerj.6722 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Goode et al. title: Modulation of biochemical and physiological parameters in Hordeum vulgare L. seedlings under the influence of benzyl-butyl phthalate link: https://peerj.com/articles/6742 last-modified: 2019-04-19 description: BackgroundPhthalates are man-made chemical compounds with numerous applications especially known for their use as plasticizers. They have weak bonding to the polymeric matrix or products in which they are used. Owing to this reason, they are readily released into the environment which makes them ubiquitous. The agricultural soils are also reported to be polluted with phthalates up to a considerable extent which causes adverse effects on flora and fauna. A few studies have been conducted on phthalate-induced phytotoxicity, which has revealed that phthalates affect the quality and yield of edible plants. In the last decades, some crops were analyzed for phthalate-induced adversities; among them, barley was the least explored.MethodsThe present study has investigated the impact of benzyl-butyl phthalate (BBP) on barley (Hordeum vulgare L.) seedlings to address the biochemical, physiological consequences, and toxicological implications. After the exogenous exposure of BBP (viz. 0, 25, 50, 100, 200, 400, 800, 1,600 mg/L) for 7 days, barley seedlings were analyzed for different indices.ResultsThe exposure of BBP mediated a significant (p ≤ 0.05, 0.01) overall elevation in the contents of pigment, proline, soluble protein, carbohydrate, hydrogen peroxide (H2O2), and malondialdehyde (MDA) in shoots and roots of barley seedlings. The activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) were also stimulated significantly in shoots and roots of seedlings against BBP stress except for SOD activity which declined in the roots. The polyphenols (non-enzymatic antioxidants) content was also altered in all the treated concentrations as compared to the control. Furthermore, BBP caused stomatal abnormalities, induced cytotoxicity, and loss of plasma membrane integrity.ConclusionsBBP disturbed the normal physiology of barley which could also affect the yield of the crop under field conditions. creator: Arpna Kumari creator: Rajinder Kaur uri: https://doi.org/10.7717/peerj.6742 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Kumari and Kaur title: Effect of irradiance on the emission of short-lived halocarbons from three common tropical marine microalgae link: https://peerj.com/articles/6758 last-modified: 2019-04-19 description: Marine algae have been reported as important sources of biogenic volatile halocarbons that are emitted into the atmosphere. These compounds are linked to destruction of the ozone layer, thus contributing to climate change. There may be mutual interactions between the halocarbon emission and the environment. In this study, the effect of irradiance on the emission of halocarbons from selected microalgae was investigated. Using controlled laboratory experiments, three tropical marine microalgae cultures, Synechococcus sp. UMACC 371 (cyanophyte), Parachlorella sp. UMACC 245 (chlorophyte) and Amphora sp. UMACC 370 (diatom) were exposed to irradiance of 0, 40 and 120 µmol photons m−2s−1. Stress in the microalgal cultures was indicated by the photosynthetic performance (Fv/Fm, maximum quantum yield). An increase in halocarbon emissions was observed at 120 µmol photons m−2s−1, together with a decrease in Fv/Fm. This was most evident in the release of CH3I by Amphora sp. Synechococcus sp. was observed to be the most affected by irradiance as shown by the increase in emissions of most halocarbons except for CHBr3 and CHBr2Cl. High positive correlation between Fv/Fm and halocarbon emission rates was observed in Synechococcus sp. for CH2Br2. No clear trends in correlation could be observed for the other halocarbons in the other two microalgal species. This suggests that other mechanisms like mitochondria respiration may contribute to halocarbon production, in addition to photosynthetic performance. creator: Yong-Kian Lim creator: Fiona Seh-Lin Keng creator: Siew-Moi Phang creator: William T. Sturges creator: Gill Malin creator: Noorsaadah Abd Rahman uri: https://doi.org/10.7717/peerj.6758 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Lim et al. title: An affordable and automated imaging approach to acquire highly resolved individual data—an example of copepod growth in response to multiple stressors link: https://peerj.com/articles/6776 last-modified: 2019-04-19 description: Individual trait variation is essential for populations to cope with multiple stressors and continuously changing environments. The immense number of possible stressor combinations and the influence of phenotypic variation makes experimental testing for effects on organisms challenging. The acquisition of such data requires many replicates and is notoriously laborious. It is further complicated when responses occur over short time periods. To overcome such challenges, we developed an automated imaging platform to acquire temporally highly resolved individual data. We tested this platform by exposing copepods to a combination of a biotic stressor (predator cues) and a toxicant (copper) and measured the growth response of individual copepods. We tested the automatically acquired data against published manually acquired data with much lower temporal resolution. We find the same general potentiating effects of predator cues on the adverse effects of copper, and the influence of an individual’s clutch identity on its ability to resist stress, between the data obtained from low and high temporal resolution. However, when using the high temporal resolution, we also uncovered effects of clutch ID on the timing and duration of stage transitions, which highlights the importance of considering phenotypic variation in ecotoxicological testing. Phenotypic variation is usually not acknowledged in ecotoxicological testing. Our approach is scalable, affordable, and adjustable to accommodate both aquatic and terrestrial organisms, and a wide range of visually detectable endpoints. We discuss future extensions that would further widen its applicability. creator: Jan Heuschele creator: Torben Lode creator: Tom Andersen creator: Katrine Borgå creator: Josefin Titelman uri: https://doi.org/10.7717/peerj.6776 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Heuschele et al. title: Using Ipomoea aquatic as an environmental-friendly alternative to Elodea nuttallii for the aquaculture of Chinese mitten crab link: https://peerj.com/articles/6785 last-modified: 2019-04-19 description: Elodea nuttallii is widely used in Chinese mitten crab (CMC) rearing practice, but it is not a native aquatic plant and cannot endure high temperature. Thus, large E. nuttallii mortality and water deterioration events could occur during high-temperature seasons. The aim of this study was to identify the use of local macrophytes in CMC rearing practice, including Ipomoea aquatic and Oryza sativa. A completely randomized field experiment was conducted to investigate the crab yield, water quality, bacterioplankton community and functions in the three different systems (E. nuttallii, I. aquatic, and O. sativa). Average crab yields in the different macrophyte systems did not differ significantly. The I. aquatic and O. sativa systems significantly decreased the total nitrogen and nitrate-N quantities in the outflow waters during the rearing period compared to the E. nuttallii system, and the I. aquatic and O. sativa plants assimilated more nitrogen than the E. nuttallii plant. Moreover, the significant changes of bacterioplankton abundances and biodiversity in the three systems implied that cleanliness of rearing waters was concomitantly attributed to the differential microbial community and functions. In addition, principle component analysis successfully differentiated the bacterioplankton communities of the three macrophytes systems. Environmental factor fitting and the co-occurrence network analyses indicated that pH was the driver of bacterioplankton community structure. Functional predictions using PICRUSt (v.1.1.3) software based on evolutionary modeling indicated a higher potential for microbial denitrification in the I. aquatic and O. sativa systems. Notably, the O. sativa plants stopped growing in the middle of the rearing period. Thus, the I. aquatic system rather than the O. sativa system could be a feasible and environmental-friendly alternative to the E. nuttallii system in CMC rearing practice. creator: Linlin Shi creator: Meijuan Jin creator: Mingxing Shen creator: Changying Lu creator: Haihou Wang creator: Xingwei Zhou creator: Lijuan Mei creator: Shixue Yin uri: https://doi.org/10.7717/peerj.6785 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Shi et al. title: Habitat suitability and connectivity inform a co-management policy of protected area network for Asian elephants in China link: https://peerj.com/articles/6791 last-modified: 2019-04-19 description: Enlarging protected area networks (PANs) is critical to ensure the long-term population viability of Asian elephants (Elephas maximus), which are threatened by habitat loss and fragmentation. Strict policies of PAN enlargement that focus on wildlife conservation have failed largely due to difficulties in encouraging stakeholder participation and meeting the elephant habitat requirement. A co-management policy that promotes sustainable resource use, wildlife conservation, and stakeholder participation may have greater feasibility than the strict policies in a developing world. Here, we identified the suitable habitat of elephants using maximum entropy models and examined whether habitat suitability is indirectly associated with local economic development in human-dominated landscapes. We found that (1) the suitable habitat was mainly in areas of forest matrix (50% natural forest cover) with multiple land-use practices rather than relatively intact forest and near communities (mean distance two km) and (2) habitat suitability was negatively associated with local economic development (rP = −0.37, P = 0.04). From the standpoint of elephant habitat and its socio-economic background, our results indicate that co-management will be more effective than the currently strict approaches of enlarging PAN. Additionally, our results provide on-ground information for elephant corridor design in southern China. creator: Cheng Huang creator: Xueyou Li creator: Laxman Khanal creator: Xuelong Jiang uri: https://doi.org/10.7717/peerj.6791 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Huang et al. title: Tracking neonicotinoids following their use as cotton seed treatments link: https://peerj.com/articles/6805 last-modified: 2019-04-19 description: Neonicotinoids are a leading class of insecticides on the global market, accounting for nearly 25%. They are widely used in both agricultural and residential settings. Causing neuron failure by irreversibly binding to the insect nicotinic acetylcholine receptor, neonicotinoids offer broad spectrum efficacy against a variety of pests. However, because they are non-selective with regard to insect species, there has been some concern with neonicotinoid use over threats to pollinators such as honeybees, and potential indirect effects to migratory waterfowl as a result of invertebrate prey population depletion. In order to study occurrence and fate of neonicotinoids (thiamethoxam and imidacloprid), we analyzed cotton leaves on plants grown from neonicotinoid-treated seeds and corresponding soil samples between cotton rows. Neonicotinoid concentration data from cotton leaves appears to be consistent with the claim that seed treatments protect plants for 3–4 weeks; by 30 days post-planting, neonicotinoid concentrations fell, in general, to 200 ng/g or lower. This represents about a 10-fold decrease from plant concentrations at approximately 2 weeks post-planting. It was found that neonicotinoids used as seed treatments remained present in the soil for months post planting and could be available for runoff. To that end, 21 playa wetlands were sampled; 10 had at least one quantifiable neonicotinoid present, three of which were classified as grassland or rangeland playas, two were urban, and the remaining five were cropland playas. In several instances, neonicotinoid concentrations in playas exceeded EPA chronic benchmarks for aquatic invertebrates. creator: Kristina L. Kohl creator: Lauren K. Harrell creator: Joseph F. Mudge creator: Seenivasan Subbiah creator: John Kasumba creator: Etem Osma creator: Apurba K. Barman creator: Todd A. Anderson uri: https://doi.org/10.7717/peerj.6805 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Kohl et al. title: Evaluation of the extent of damage to the esophageal wall caused by press-through package ingestion link: https://peerj.com/articles/6763 last-modified: 2019-04-18 description: Press-through package (PTP) is the most common accidentally ingested foreign body in Japan. Accidental ingestion of PTP can result in esophageal damage. An approach for evaluating the risk of esophageal injury has not been established. Therefore, we used porcine esophageal tissue and silicone sheets to establish a method for assessing the risk of esophageal damage on accidental PTP ingestion. We pathologically evaluated porcine lower esophageal tissue using a scratch tester. Using porcine esophageal tissue, scratch tests were performed with 4 test objects and pathological damage was compared. It was assumed that each object was accidentally ingested. The objects were polyvinylidene chloride (PVDC)-coated polyvinyl chloride (PVC) PTP, soft PThPa, round PTP, and a disposable scalpel. The porcine esophagus was replaced with a silicon sheet, and an automatic friction machine was used for quantitative evaluation. The silicon sheet was scratched using HHS 2000 with 750-g load at 50 mm/min. We investigated the frictional force exerted on the surface for each of the objects. The degree of damage (depth) was the highest for the disposable scalpel, followed by PVDC-coated PVC PTP, while the degree of damage (depth) was the lowest for soft PThPa and round PTP. The mean frictional forces on the silicon sheet were 524.0 gf with PVDC-coated PTP, 323.5 gf with soft PThPa, 288.7 gf with round PTP, and 922.7 gf with the disposable scalpel. We developed approaches to qualitatively and quantitatively evaluate the risk of esophageal damage after accidental PTP ingestion. Our findings indicate that the risk of gastrointestinal damage after accidental PTP ingestion is low with soft PTP and round PTP. creator: Takuya Tamura creator: Hajime Okamoto creator: Toyoaki Suzuki creator: Yoichi Nakanishi creator: Daisuke Sugiyama uri: https://doi.org/10.7717/peerj.6763 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Tamura et al. title: Bacterial community composition and diversity of two different forms of an organic residue of bioenergy crop link: https://peerj.com/articles/6768 last-modified: 2019-04-18 description: The use of residue of sugarcane ethanol industry named vinasse in fertirrigation is an established and widespread practice in Brazil. Both non-concentrated vinasse (NCV) and concentrated vinasse (CV) are used in fertirrigation, particularly to replace the potassium fertilizer. Although studies on the chemical and organic composition of vinasse and their impact on nitrous oxide emissions when applied in soil have been carried out, no studies have evaluated the microbial community composition and diversity in different forms of vinasse. We assessed the bacterial community composition of NCV and CV by non-culturable and culturable approaches. The non-culturable bacterial community was assessed by next generation sequencing of the 16S rRNA gene and culturable community by isolation of bacterial strains and molecular and biochemical characterization. Additionally, we assessed in the bacterial strains the presence of genes of nitrogen cycle nitrification and denitrification pathways. The microbial community based on 16S rRNA sequences of NCV was overrepresented by Bacilli and Negativicutes while CV was mainly represented by Bacilli class. The isolated strains from the two types of vinasse belong to class Bacilli, similar to Lysinibacillus, encode for nirK gene related to denitrification pathway. This study highlights the bacterial microbial composition particularly in CV what residue is currently recycled and recommended as a sustainable practice in sugarcane cultivation in the tropics. creator: Matheus A.P. Cipriano creator: Afnan K.A. Suleiman creator: Adriana P.D. da Silveira creator: Janaína B. do Carmo creator: Eiko E. Kuramae uri: https://doi.org/10.7717/peerj.6768 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Cipriano et al. title: Bioeffects of Prunus spinosa L. fruit ethanol extract on reproduction and phenotypic plasticity of Trichoplax adhaerens Schulze, 1883 (Placozoa) link: https://peerj.com/articles/6789 last-modified: 2019-04-18 description: The aim of this work was to test and analyse the bioeffects of Prunus spinosa L. (Rosacaee) fruit ethanol extract on Trichoplax adhaerens Schulze, 1883 (Placozoa) laboratory cultures which—for the first time—were employed as in vivo biological model to assess the bioactivity of a natural extract. The ethanol extract of P. spinosa was administrated during a 46 day experimental period; ultrastructural (by optical, confocal, TEM and SEM microscopy) and morphometric analyses indicated that treated Trichoplax adhaerens showed significant differences in viability, reproductive modalities, body shape and colour with respect to the control group. Finally, P. spinosa bioactive compounds seem to exert profound protective effects on T. adhaerens reproduction and phenotype. Our results may support additional investigations related to other bioactive compounds properties useful for nutraceutical preparations to be used as food supplements. creator: Maria Cristina Albertini creator: Daniele Fraternale creator: Federica Semprucci creator: Silvio Cecchini creator: Mariastella Colomba creator: Marco B.L. Rocchi creator: Davide Sisti creator: Barbara Di Giacomo creator: Michele Mari creator: Luigia Sabatini creator: Lucia Cesaroni creator: Maria Balsamo creator: Loretta Guidi uri: https://doi.org/10.7717/peerj.6789 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Albertini et al. title: Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber–pumpkin grafted plants link: https://peerj.com/articles/6536 last-modified: 2019-04-17 description: BackgroundQuantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants.MethodsIn this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder.ResultsThe results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting.DiscussionThis work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants. creator: Li Miao creator: Xing Qin creator: Lihong Gao creator: Qing Li creator: Shuzhen Li creator: Chaoxing He creator: Yansu Li creator: Xianchang Yu uri: https://doi.org/10.7717/peerj.6536 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Miao et al. title: The coast of giants: an anthropometric survey of high schoolers on the Adriatic coast of Croatia link: https://peerj.com/articles/6598 last-modified: 2019-04-17 description: The aim of this anthropometric survey was to map regional differences in height and body proportions in eight counties adjacent to the Adriatic coast of Croatia. Body height was measured in 1,803 males and 782 females aged 17–20 years at 66 schools in 23 towns. When corrected for population size in regions, mean male height is 182.6 cm in all eight counties, 182.8 cm in seven counties of Adriatic Croatia, and 183.7 cm in four counties of Dalmatia proper. Regional variation is considerable: from 180.6 cm in the county of Karlovac to 184.1 cm in the county of Split-Dalmacija. The mean height of females is based on more limited data (168.0 cm in seven counties). These results show that young men from Dalmatia are currently the tallest in the world in the age category of 18 years, and the north-to-south gradient of increasing stature on the Adriatic coast largely mirrors that in neighbouring Bosnia and Herzegovina (BiH). The extraordinary values of height in Croatia and BiH can most likely be explained by unique genetic predispositions that are shared by the local populations of the Dinaric Alps. creator: Pavel Grasgruber creator: Stipan Prce creator: Nikola Stračárová creator: Eduard Hrazdíra creator: Jan Cacek creator: Stevo Popović creator: Sylva Hřebíčková creator: Predrag Potpara creator: Ivan Davidovič creator: Tomáš Kalina uri: https://doi.org/10.7717/peerj.6598 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Grasgruber et al. title: Boto (Inia geoffrensis—Cetacea: Iniidae) aggregations in two provisioning sites in the lower Negro River—Amazonas, Brazil: are they related? link: https://peerj.com/articles/6692 last-modified: 2019-04-17 description: The Negro River currently has seven floating houses where tourists can feed and interact with botos, each with its own history of how these aggregations were formed. Some keepers say these groups are familial, even reporting individuals being born into the group. However, behavioral studies have shown that botos are solitary, only forming groups at feeding areas and during the mating season. In the present study we used 12 microsatellite and molecular sex markers to characterize relationships within and between two boto aggregations (ten and seven botos each) in the lower Negro River. Molecular sexing revealed that all botos sampled from both aggregations were males. This may be explained by habitat preference, as male botos are primarily found in the main channels of large rivers, whereas females prefer more protected areas, such as flooded forests and its channels and lakes. Most of the animals were unrelated within each aggregation, demonstrating that these aggregations are not normally formed due to kinship bonds, but are exclusively for feeding, as botos learn that these places provide easy access to food. This study provides important information that helps us understand how human interaction is affecting the social structure and behavior of these animals. creator: Waleska Gravena creator: Tomas Hrbek creator: Vera Maria Ferreira da Silva creator: Izeni Pires Farias uri: https://doi.org/10.7717/peerj.6692 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Gravena et al. title: Validation of Plasmodium falciparum deoxyhypusine synthase as an antimalarial target link: https://peerj.com/articles/6713 last-modified: 2019-04-17 description: BackgroundHypusination is an essential post-translational modification in eukaryotes. The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved. Plasmodium falciparum human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs.MethodsTransgenic P. falciparum parasites with modification of the PF3D7_1412600 gene encoding PfDHS enzyme were created by insertion of the glmS riboswitch or the M9 inactive variant. The PfDHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting. The biochemical function of PfDHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays. Gene essentiality was assessed by competitive growth assays and chemogenomic profiling.ResultsClonal transgenic parasites with integration of glmS riboswitch downstream of the PfDHS gene were established. PfDHS protein was present in the cytoplasm of transgenic parasites in asexual stages. The PfDHS protein could be attenuated fivefold in transgenic parasites with an active riboswitch, whereas PfDHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence. Attenuation of PfDHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected. Parasites with attenuated PfDHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) expression. PfDHS-attenuated parasites showed increased sensitivity to N1-guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes.DiscussionLoss of PfDHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins. The growth defect in parasites with attenuated Pf DHS expression suggests that this gene is essential. However, the slower decline of PfDHS mutants compared with PfDHFR-TS mutants in competitive growth assays suggests that PfDHS is less vulnerable as an antimalarial target. Nevertheless, the data validate PfDHS as an antimalarial target which can be inhibited by spermidine-like compounds. creator: Aiyada Aroonsri creator: Navaporn Posayapisit creator: Jindaporn Kongsee creator: Onsiri Siripan creator: Danoo Vitsupakorn creator: Sugunya Utaida creator: Chairat Uthaipibull creator: Sumalee Kamchonwongpaisan creator: Philip J. Shaw uri: https://doi.org/10.7717/peerj.6713 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Aroonsri et al. title: Comparisons of WUE in twelve genotypes of winter wheat and the relationship between δ13C and WUE link: https://peerj.com/articles/6767 last-modified: 2019-04-17 description: Twelve winter wheat (Triticum aestivum) genotypes were examined for differences in grain yield, water use efficiency (WUE), and stable carbon isotope composition (δ13C) in flag leaves. The plants were subjected to rain-fed treatment and supplemental irrigation at the jointing and anthesis stages, during the 2015–2016 and 2016–2017 winter wheat growing seasons. The relationships between δ13C with grain yield and WUE were analyzed under two different water environments. The results indicated that there were significant differences in δ13C, grain yield, and WUE among wheat genotypes both under rain-fed and supplemental irrigation conditions. The δ13C values increased with grain-filling proceeding, the δ13C being lower under supplemental irrigation treatment than that under rain-fed treatment. The relationships between the average of δ13C with grain yield and WUE were significantly positive during three measurement periods (R2 = 0.5785 − 0.8258), whether under rain-fed or irrigation environments. This suggests that δ13C might be associated with the grain yield and WUE in winter wheat under rain-fed and supplemental irrigation conditions in the climate region of the northwest Huang-Huai-Hai Plain of China. creator: Guirong Huang creator: Xinying Zhang creator: Yajing Wang creator: Fu Feng creator: Xurong Mei creator: Xiuli Zhong uri: https://doi.org/10.7717/peerj.6767 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Huang et al. title: Understanding the NaCl-dependent behavior of hydrogen production of a marine bacterium, Vibrio tritonius link: https://peerj.com/articles/6769 last-modified: 2019-04-17 description: Biohydrogen is one of the most suitable clean energy sources for sustaining a fossil fuel independent society. The use of both land and ocean bioresources as feedstocks show great potential in maximizing biohydrogen production, but sodium ion is one of the main obstacles in efficient bacterial biohydrogen production. Vibrio tritonius strain AM2 can perform efficient hydrogen production with a molar yield of 1.7 mol H2/mol mannitol, which corresponds to 85% theoretical molar yield of H2 production, under saline conditions. With a view to maximizing the hydrogen production using marine biomass, it is important to accumulate knowledge on the effects of salts on the hydrogen production kinetics. Here, we show the kinetics in batch hydrogen production of V. tritonius strain AM2 to investigate the response to various NaCl concentrations. The modified Han–Levenspiel model reveals that salt inhibition in hydrogen production using V. tritonius starts precisely at the point where 10.2 g/L of NaCl is added, and is critically inhibited at 46 g/L. NaCl concentration greatly affects the substrate consumption which in turn affects both growth and hydrogen production. The NaCl-dependent behavior of fermentative hydrogen production of V. tritonius compared to that of Escherichia coli JCM 1649 reveals the marine-adapted fermentative hydrogen production system in V. tritonius. V. tritonius AM2 is capable of producing hydrogen from seaweed carbohydrate under a wide range of NaCl concentrations (5 to 46 g/L). The optimal salt concentration producing the highest levels of hydrogen, optimal substrate consumption and highest molar hydrogen yield is at 10 g/L NaCl (1.0% (w/v)). creator: Nurhidayu Al-saari creator: Eri Amada creator: Yuta Matsumura creator: Mami Tanaka creator: Sayaka Mino creator: Tomoo Sawabe uri: https://doi.org/10.7717/peerj.6769 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Al-saari et al. title: Patient-reported fatigue in patients with rheumatoid arthritis who commence biologic therapy: a longitudinal study link: https://peerj.com/articles/6771 last-modified: 2019-04-17 description: Aims and objectivesTo examine changes in patient-reported fatigue, over a twelve month period, in rheumatoid arthritis patients who commence biologic treatment, and to identify possible predictors for such changes.BackgroundFatigue is a burdensome symptom for patients with rheumatoid arthritis. Despite biologics being effective in reducing disease activity, patients still report fatigue.DesignA longitudinal observational study.MethodsA total of 48 patients were enrolled in the study. Fatigue was measured by the Fatigue Severity Scale. Independent samples T-tests were used to test gender differences, and paired samples T-tests were used to measure differences between repeated measures. Bivariate and multiple regression analyses were used to examine potential predictors for changes in fatigue, such as age, sex, Disease Activity Score 28, pain and physical and emotional well-being.ResultsForty-seven patients completed the study. From baseline to 12-month follow-up, fatigue decreased significantly in both women and men. Analyses of predictors were performed step-wise, and the final model included sex and physical well-being. The results from this final step showed that female sex was the only significant predictor for changes in fatigue.ConclusionPatients commencing biologic therapy reported a significant reduction in fatigue. Female sex was a significant predictor of changes in fatigue.Relevance to clinical practiceDespite improvements in pharmacological treatment, patients with rheumatoid arthritis still report fatigue. This is a multifaceted health problem encompassing personal and emotional factors in addition to the clinical factors directly connected to the disease. creator: Hege Selheim Rinke creator: Clara Beate Gram Gjesdal creator: Heidi Markussen creator: Jörg Assmus creator: Gerd Karin Natvig uri: https://doi.org/10.7717/peerj.6771 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Rinke et al. title: Molecular characterization and phylogenetic analysis of a Squash leaf curl virus isolate from Baja California Sur, Mexico link: https://peerj.com/articles/6774 last-modified: 2019-04-17 description: BackgroundThe begomovirus, squash leaf curl virus (SLCuV) is one of the causal agents of squash leaf curl (SLC) disease, which is among the most destructive diseases of cucurbit crops in tropical, subtropical, and semiarid regions worldwide. This disease was originally reported in the American continent with subsequent spread to the Mediterranean basin. Up to now, SLCuV has only been detected by PCR in Mexico. This study provides the first complete sequence of a Mexican SLCuV isolate from Baja California Sur (BCS). In addition, the genome of the virus was characterized, establishing its phylogenetic relationship with other SLCuV isolates.MethodsThe full genome (DNA-A and DNA-B) was amplified by rolling circle amplification, cloned and sequenced and the open reading frames (ORF) were annotated. Virus identification was performed according to the International Committee on Taxonomy of Viruses (ICTV) criteria for begomovirus species demarcation. To infer evolutionary relationship with other SLCuV isolates, phylogenetic and recombination analyses were performed.ResultsThe SLCuV-[MX-BCS-La Paz-16] genome (DNA-A and DNA-B) had 99% identity with SLCuV reference genomes. The phylogenetic analysis showed that SLCuV-[MX-BCS-La Paz-16] is closely related to SLCuV isolates from the Middle East (Egypt, Israel, Palestine and Lebanon). No evidence of interspecific recombination was determined and iterons were 100% identical in all isolates in the SLCuV clade.ConclusionsSLCuV-[MX-BCS-La Paz-16] showed low genetic variability in its genome, which could be due to a local adaptation process (isolate environment), suggesting that SLCuV isolates from the Middle East could have derived from the southwestern United States of America (USA) and northwestern Mexico. creator: Diana Medina-Hernández creator: M. Goretty Caamal-Chan creator: Mayela Vargas-Salinas creator: Abraham Loera-Muro creator: Aarón Barraza creator: Ramón Jaime Holguín-Peña uri: https://doi.org/10.7717/peerj.6774 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Medina-Hernández et al. title: Assessment on latitudinal tree species richness using environmental factors in the southeastern United States link: https://peerj.com/articles/6781 last-modified: 2019-04-17 description: The southeastern region of the United States exhibits an unusual trend of decreasing tree species richness (TSR) from higher to lower latitudes over the Florida peninsula. This trend contradicts the widely marked latitudinal diversity gradient where species richness is highest in tropical zones and decreases towards extratropical regions. This study aims to assess the environmental factors that prompt this atypical inverse latitudinal gradient seen in TSR using the USDA Forest Service’s Forest Inventory and Analysis (FIA) database. Fifteen variables under four categories of forested area, groundwater, soil properties, and climate groups were examined to model TSR in the region. Generalized linear models (GLMs) with Poisson distributions first assessed individual variables to test explanatory power then the LASSO regularization method was utilized to extract two subsets of the most influential variables to predict TSR. Forest area and four climate variables (mean annual temperature, precipitation seasonality, mean temperature of coldest quarter, and mean precipitation of driest quarter) were the top five variables during the initial GLM assessment implying their potential individual influence in regulating TSR. Two subsets of LASSO models contained seven and three predictor variables, respectively. Frist subset includes seven predictors, presented in highest to low standardized coefficient, mean temperature of coldest quarter, forested area, precipitation seasonality, mean precipitation of driest quarter, water table depth, spodosol, and available water storage. The other subset further excluded four lowest influential variables from the first set, leaving the top three variables from the first subset. The first subset of the LASSO model predicted TSR with 63.4% explained deviance while the second subset reproduced 60.2% of deviance explained. With only three variables used, the second model outperformed the first model evaluated by the AIC value. We conclude that forest patch area, mean temperature of coldest quarter, and precipitation seasonality are the highly influential variables of TSR among environmental factors in the southeastern region of U.S., but evolutionary or historic cause should be further incorporated to fully understand tree species diversity pattern in this region. creator: Youngsang Kwon creator: Taesoo Lee creator: Alison Lang creator: Dorian Burnette uri: https://doi.org/10.7717/peerj.6781 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Kwon et al. title: The synergistic effect of concatenation in phylogenomics: the case in Pantoea link: https://peerj.com/articles/6698 last-modified: 2019-04-16 description: With the increased availability of genome sequences for bacteria, it has become routine practice to construct genome-based phylogenies. These phylogenies have formed the basis for various taxonomic decisions, especially for resolving problematic relationships between taxa. Despite the popularity of concatenating shared genes to obtain well-supported phylogenies, various issues regarding this combined-evidence approach have been raised. These include the introduction of phylogenetic error into datasets, as well as incongruence due to organism-level evolutionary processes, particularly horizontal gene transfer and incomplete lineage sorting. Because of the huge effect that this could have on phylogenies, we evaluated the impact of phylogenetic conflict caused by organism-level evolutionary processes on the established species phylogeny for Pantoea, a member of the Enterobacterales. We explored the presence and distribution of phylogenetic conflict at the gene partition and nucleotide levels, by identifying putative inter-lineage recombination events that might have contributed to such conflict. Furthermore, we determined whether smaller, randomly constructed datasets had sufficient signal to reconstruct the current species tree hypothesis or if they would be overshadowed by phylogenetic incongruence. We found that no individual gene tree was fully congruent with the species phylogeny of Pantoea, although many of the expected nodes were supported by various individual genes across the genome. Evidence of recombination was found across all lineages within Pantoea, and provides support for organism-level evolutionary processes as a potential source of phylogenetic conflict. The phylogenetic signal from at least 70 random genes recovered robust, well-supported phylogenies for the backbone and most species relationships of Pantoea, and was unaffected by phylogenetic conflict within the dataset. Furthermore, despite providing limited resolution among taxa at the level of single gene trees, concatenated analyses of genes that were identified as having no signal resulted in a phylogeny that resembled the species phylogeny of Pantoea. This distribution of signal and noise across the genome presents the ideal situation for phylogenetic inference, as the topology from a ≥70-gene concatenated species phylogeny is not driven by single genes, and our data suggests that this finding may also hold true for smaller datasets. We thus argue that, by using a concatenation-based approach in phylogenomics, one can obtain robust phylogenies due to the synergistic effect of the combined signal obtained from multiple genes. creator: Marike Palmer creator: Stephanus N. Venter creator: Alistair R. McTaggart creator: Martin P.A. Coetzee creator: Stephanie Van Wyk creator: Juanita R. Avontuur creator: Chrizelle W. Beukes creator: Gerda Fourie creator: Quentin C. Santana creator: Magriet A. Van Der Nest creator: Jochen Blom creator: Emma T. Steenkamp uri: https://doi.org/10.7717/peerj.6698 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Palmer et al. title: Cloning and characterization of a tyrosine decarboxylase involved in the biosynthesis of galanthamine in Lycoris aurea link: https://peerj.com/articles/6729 last-modified: 2019-04-16 description: BackgroundGalanthamine, one kind of Amaryllidaceae alkaloid extracted from the Lycoris species, is used in the treatment of Alzheimer’s disease. In regards to medical and economic importance, the biosynthesis and regulatory mechanism of the secondary metabolites in Lycoris remain uninvestigated.MethodsBLAST was used to identify the sequence of tyrosine decarboxylase in the transcriptome of Lycoris aurea (L’Hér) Herb. The enzyme activity of this TYDC was determined by using heterologous expressed protein in the Escherichia coli cells. The related productive contents of tyramine were detected using High Performance Liquid Chromatography (HPLC). According to the available micro RNA sequencing profiles and degradome database of L. aurea, microRNA396 were isolated, which targets to LaTYDC1 and RNA Ligase-Mediated-Rapid Amplification of cDNA Ends (RLM-RACE) were used to confirm the cleavage. The expression levels of miR396 and LaTYDC1 were measured using a quantitative real-time polymerase chain reaction (qRT-PCR).ResultsLaTYDC1 was mainly expressed in root, bulb, leaf and flower fitting the models for galanthamine accumulation. This decarboxylase efficiently catalyzes tyrosine to tyramine conversion. Under methyl jasmonate (MeJA) treatment, the expression of LaTYDC1 and the content of tyramine sharply increase. The use of RLM-RACE confirms that miR396 promotes the degradation of LaTYDC1 mRNA. Under MeJA treatment, the expression of miR396 was suppressed while the expression level of LaTYDC1 sharply increased. Following the increase of the miR396 transcriptional level, LaTYDC1 was significantly repressed.ConclusionLaTYDC1 participates in the biosynthesis of galanthamine, and is regulated by miR396. This finding also provides genetic strategy for improving the yield of galanthamine in the future. creator: Rong Wang creator: Xiaokang Han creator: Sheng Xu creator: Bing Xia creator: Yumei Jiang creator: Yong Xue creator: Ren Wang uri: https://doi.org/10.7717/peerj.6729 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Wang et al. title: Simulating the effects of climate change across the geographical distribution of two medicinal plants in the genus Nardostachys link: https://peerj.com/articles/6730 last-modified: 2019-04-16 description: BackgroundThe medicinal plants of Radix et Rhizoma Nardostachyos include Nardostachys jatamansi and N. chinensis. Traditionally, the two plants have been used to treat many diseases. Because of their special aroma, they are also commonly used in the food and cosmetics industry. Recently, N. jatamansi and N. chinensis have been overexploited due to their economic importance, resulting in a sharp decline in their wild resources. Predicting potential distributions of the genus Nardostachys under different climate scenarios and understanding its preferred habitat are of great significance for their conservation, artificial cultivation, and assessment of their value.MethodsThe Maxent model was used to predict the potential geographical distributions of the genus Nardostachys under current and future climatic conditions based on two representative concentration pathways (RCP2.6 and RCP8.5) for the 2050s and 2070s. These data were used to study the effects of climate variables.ResultsThe results show that the potential distribution of the two species will increase, thus more suitable habitats will be present in China. The suitable habitat for N. chinensis presents a relatively stable growth compared to N. jatamansi. In addition, precipitation plays a crucial role in modeling the effects of climate change on the genus Nardostachys. This study provides theoretical guidance for the cultivation of N. chinensis. creator: Junjun Li creator: Jie Wu creator: Kezhong Peng creator: Gang Fan creator: Haiqing Yu creator: Wenguo Wang creator: Yang He uri: https://doi.org/10.7717/peerj.6730 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Li et al. title: Characterization of the iPSC-derived conditioned medium that promotes the growth of bovine corneal endothelial cells link: https://peerj.com/articles/6734 last-modified: 2019-04-16 description: Corneal endothelial cells (CECs) maintain corneal transparency and visual acuity. However, the limited proliferative capability of these cells in vitro has prompted researchers to find efficient culturing techniques for them. The aim of our study was to evaluate the use of conditioned medium (CM) obtained from induced pluripotent stem cells (iPSCs) as a source for the effective proliferation of bovine CECs (B-CECs). In our study, the proliferative ability of B-CECs was moderately enhanced when the cells were grown in 25% iPSC conditioned medium (iPSC-CM). Additionally, hexagonal cell morphology was maintained until passage 4, as opposed to the irregular and enlarged shape observed in control corneal endothelial medium (CEM). B-CECs in both the 25% iPSC-CM and CEM groups expressed and Na+-K+-ATPase. The gene expression levels of NIFK, Na+-K+-ATPase, Col4A and Col8A and the percentage of cells entering S and G2 phases were higher in the iPSC-CM group. The number of apoptotic cells also decreased in the iPSC-CM group. In comparison to the control cultures, iPSC-CM facilitated cell migration, and these cells showed better barrier functions after several passages. The mechanism of cell proliferation mediated by iPSC-CM was also investigated, and phosphorylation of Akt was observed in B-CECs after exposure to iPSC-CM and showed sustained phosphorylation induced for up to 180 min in iPSC-CM. Our findings indicate that iPSC-CM may employ PI3-kinase signaling in regulating cell cycle progression, which can lead to enhanced cellular proliferation. Effective component analysis of the CM showed that in the iPSC-CM group, the expression of activin-A was significantly increased. If activin-A is added as a supplement, it could help to maintain the morphology of the cells, similar to that of CM. Hence, we conclude that activin-A is one of the effective components of CM in promoting cell proliferation and maintaining cell morphology. creator: Qing Liu creator: Yonglong Guo creator: Shiwei Liu creator: Peiyuan Wang creator: Yunxia Xue creator: Zekai Cui creator: Jiansu Chen uri: https://doi.org/10.7717/peerj.6734 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Liu et al. title: Identification and expression analysis of the GDSL esterase/lipase family genes, and the characterization of SaGLIP8 in Sedum alfredii Hance under cadmium stress link: https://peerj.com/articles/6741 last-modified: 2019-04-16 description: BackgroundThe herb Sedum alfredii (S. alfredii) Hance is a hyperaccumulator of heavy metals (cadmium (Cd), zinc (Zn) and lead (Pb)); therefore, it could be a candidate plant for efficient phytoremediation. The GDSL esterase/lipase protein (GELP) family plays important roles in plant defense and growth. Although the GELP family members in a variety of plants have been cloned and analyzed, there are limited studies on the family’s responses to heavy metal-stress conditions.MethodsMultiple sequence alignments and phylogenetic analyses were performed according to the criteria described. A WGCNA was used to construct co-expression regulatory networks. The roots of S. alfredii seedlings were treated with 100 µM CdCl2 for qRT-PCR to analyze expression levels in different tissues. SaGLIP8 was transformed into the Cd sensitive mutant strain yeast Δycf1 to investigate its role in resistance and accumulation to Cd.ResultsWe analyzed GELP family members from genomic data of S. alfredii. A phylogenetic tree divided the 80 identified family members into three clades. The promoters of the 80 genes contained certain elements related to abiotic stress, such as TC-rich repeats (defense and stress responsiveness), heat shock elements (heat stress) and MYB-binding sites (drought-inducibility). In addition, 66 members had tissue-specific expression patterns and significant responses to Cd stress. In total, 13 hub genes were obtained, based on an existing S. alfredii transcriptome database, that control 459 edge genes, which were classified into five classes of functions in a co-expression subnetwork: cell wall and defense function, lipid and esterase, stress and tolerance, transport and transcription factor activity. Among the hub genes, Sa13F.102 (SaGLIP8), with a high expression level in all tissues, could increase Cd tolerance and accumulation in yeast when overexpressed.ConclusionBased on genomic data of S. alfredii, we conducted phylogenetic analyses, as well as conserved domain, motif and expression profiling of the GELP family under Cd-stress conditions. SaGLIP8 could increase Cd tolerance and accumulation in yeast. These results indicated the roles of GELPs in plant responses to heavy metal exposure and provides a theoretical basis for further studies of the SaGELP family’s functions. creator: He Li creator: Xiaojiao Han creator: Wenmin Qiu creator: Dong Xu creator: Ying Wang creator: Miao Yu creator: Xianqi Hu creator: Renying Zhuo uri: https://doi.org/10.7717/peerj.6741 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Li et al. title: Risk of acute exacerbation between acetaminophen and ibuprofen in children with asthma link: https://peerj.com/articles/6760 last-modified: 2019-04-16 description: BackgroundAntipyretics are widely prescribed in pediatric practice. Some reports have mentioned that acetaminophen and non-steroid anti-inflammatory drugs may negatively affect asthma control by causing asthma exacerbation (AE). However, many confounding factors can also influence the risks. We assessed the impact of using acetaminophen or ibuprofen on AE in asthmatic children, especially those with strong risk factors.MethodsWe used the 2010 Taiwan National Health Insurance Research Database and identified 983 children with persistent asthma aged 1–5 years old; among them, 591 used acetaminophen alone and 392 used ibuprofen alone in 2010. Then, we analyzed the risk of AE over 52 weeks in the patients with and without severe AE in the previous year.ResultsThe ibuprofen group had a higher risk of an emergency room (ER) visit or hospitalization for AE (odds ratio (OR) = 2.10, 95% confidence interval (CI) [1.17–3.76], P = 0.01). Among asthmatic children who had severe AE in the previous year, the risk of AE was higher in the ibuprofen group than in the acetaminophen group (OR = 3.28, 95% CI [1.30–8.29], P = 0.01), where as among those who did not, the risks of AE were similar between the acetaminophen and ibuprofen groups (OR = 1.52, 95% CI [0.71–3.25], P = 0.28).ConclusionsAmong young asthmatic children, use of ibuprofen was associated with a higher risk of AE than acetaminophen, if they had severe AE with ER visit or hospitalization in the previous year. Pediatricians should use antipyretics among children with asthma after a full evaluation of the risk. creator: Lin-Shien Fu creator: Che-Chen Lin creator: Chia-Yi Wei creator: Ching-Heng Lin creator: Yung-Chieh Huang uri: https://doi.org/10.7717/peerj.6760 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Fu et al. title: Distribution of crabs along a habitat gradient on the Yellow Sea coast after Spartina alterniflora invasion link: https://peerj.com/articles/6775 last-modified: 2019-04-16 description: The effects of Spartina alterniflora invasion on macrobenthos have long been of concern; however, there is currently no unified conclusion regarding these effects. Most studies on crabs focus on one species or limited habitat types, and assessments of the community-level effects of S. alterniflora invasion considering multiple species and habitat types have rarely been conducted. In this study, we sampled crabs along a habitat gradient from the shoreline to inland areas on the Yellow Sea coast, including the mudflat, S. alterniflora marsh, Suaeda salsa marsh and Phragmites australis marsh. A total of 10 crab species were found among all habitats, with five species in the mudflat, six species in S. alterniflora marsh, seven species in S. salsa marsh and four species in P. australis marsh. The Shannon index values for the crab communities were similar between S. alterniflora marsh and S. salsa marsh, and these values were significantly higher than those for the mudflat and P. australis marsh. However, the total biomass of crabs was highest in the mudflat, and Metaplax longipes, Philyra pisum and Macrophthalmus dilatatus exclusively preferred the mudflat. The analysis of principal components and similarities showed that the crab community structure in S. alterniflora marsh was most similar to that in S. salsa marsh, while the crab community structure in the mudflat was most different from that in the other habitat types. Our results demonstrate that the distribution of crabs varies across a habitat gradient after S. alterniflora invasion and that the crab community in S. alterniflora marsh is slightly different from that associated with the local vegetation but shows a large difference from that in the mudflat. This study indicates that some crab species may have adapted to habitat containing alien S. alterniflora, while other crab species reject this new marsh type. The effects of the distribution of crabs after S. alterniflora invasion on the regional ecosystem need further study in the future. creator: Pan Chen creator: Yan Zhang creator: Xiaojing Zhu creator: Changhu Lu uri: https://doi.org/10.7717/peerj.6775 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Chen et al. title: Informing the management of acute malnutrition in infants aged under 6 months (MAMI): risk factor analysis using nationally-representative demographic & health survey secondary data link: https://peerj.com/articles/5848 last-modified: 2019-04-15 description: BackgroundTackling malnutrition is a global health priority, helping children both survive and thrive. Acute malnutrition (wasting) in infants aged under 6 months (u6m) is often neglected. Worldwide, some 8.5 million infants u6m are affected yet recent World Health Organization malnutrition guidelines highlight numerous evidence gaps on how to best manage them. To inform future research, policy and programming, we aimed to identify risk factors associated with infant u6m wasting.MethodsWe did secondary data analysis of nationally representative, cross sectional Demographic and Health Surveys conducted in the last 10 years. We compared wasted infants u6m (weight-for-length <−2 z-scores) vs. non-wasted (weight-for-length ≥−2 z-score). We used simple and adjusted (for infant age, sex, socio-economic status) logistic regression to calculate odds of wasting associated with risk factors spanning three broad categories: household-related; maternal-related; infant-related.ResultsWe analysed 16,123 infants u6m from 20 countries. Multiple risk factors were statistically associated with wasting. These included: poverty (Odds ratio, OR 1.22 (95% CI [1.01–1.48], p = 0.04)); low maternal body mass index (adjusted OR 1.53(1.29–1.80, p < 0.001); small infant size at birth (aOR 1.32(1.10–1.58, p < 0.01)); delayed start of breastfeeding (aOR 1.31(1.13–1.51, p < 0.001)); prelacteal feed (aOR 1.34(1.18–1.53, p < 0.001)); recent history of diarrhoea (aOR 1.37(1.12–1.67, p < 0.01)); mother disempowered (experiences violence; does not make decisions about health issues; does not engage with health services such as antenatal care, does not give birth in a health facility). ‘Protective’ factors associated with significantly decreased odds of infant u6m wasting included: educated mother (OR 0.64(0.54–0.76, p < 0.001)); mother in work (OR 0.82(0.72–0.94, p < 0.01)); currently breastfed (aOR 0.62(0.42–0.91, p = 0.02)), exclusively breastfed (aOR 0.84(0.73–0.97, p = 0.02).DiscussionInfant u6m wasting is a complex, multifactorial problem associated with many risk factors; knowing them will help shape international and national management strategies. Whilst our observational study cannot prove causation, many factors identified are biologically plausible and/or socially important. They should be considered when assessing and managing infants u6m. Although supporting breastfeeding is core to future interventions, this alone is unlikely to be sufficient; strategies should involve multiple sectors, beyond just health and nutrition. By noting our results, future intervention studies could focus resources and maximise chances of achieving impact. creator: Marko Kerac creator: Severine Frison creator: Nichola Connell creator: Bethan Page creator: Marie McGrath uri: https://doi.org/10.7717/peerj.5848 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Kerac et al. title: No association of COMT with insight problem solving in Chinese college students link: https://peerj.com/articles/6755 last-modified: 2019-04-15 description: Genes involved in dopamine (DA) neurotransmission, such as the catechol-O-methyltransferase gene (COMT), have been suggested as key genetic candidates that might underlie the genetic basis of insight. In a sample of Chinese college students, this study examined whether COMT was associated with individual differences in the ability to solve classic insight problems. The results demonstrated that COMT was not associated with insight problem solving and there was no gender-dependent effect. This study, together with previous studies, raises the possibility of a complex relationship between COMT and insight problem solving. creator: Xiaolei Yang creator: Jinghuan Zhang creator: Shun Zhang uri: https://doi.org/10.7717/peerj.6755 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Yang et al. title: Head louse egg and nit remover—a modern “Quest for the Holy Grail” link: https://peerj.com/articles/6759 last-modified: 2019-04-15 description: BackgroundThe eggs of head lice are fixed to the hair of their hosts by means of a persistent glue-like fixative that is not chemically bound to the substrate. Eggshells stuck to hairs after successfully treating the infestation are a cosmetic issue and a source of misunderstanding about whether the infestation is eliminated. Hitherto, no effective treatment to loosen louse eggs and nits has been found.MethodsAn extensive screening of surface active compounds, oils, esters, and other cosmetic lubricants used a slip-peel device to measure the forces required to release the grip of the fixative. Promisingly effective compounds were formulated into suitable carriers for further testing. The most effective combination formulation was tested, as a commercial product (Hedrin Stubborn Egg Loosening Lotion), in a usage study of 15 children with nits, in which one half of the head was combed only on damp hair and the other half combed after a 10 min treatment using the product.ResultsLaboratory tests of the forces required to remove nits found that pelagonic acid derivatives, particularly isononyl isononanoate, in the presence of a polymeric gelling agent and water, were most effective to reduce the initial grip of the fixative as well as reducing friction as the eggshell is drawn along the hair shaft and that the final product was significantly (p < 0.05) more effective than several other marketed materials. In the usage study significantly (p = 0.01046) more louse eggs and nits were removed after treatment with the gel.DiscussionThe product developed through this study is the first with a demonstrable efficacy for loosening the grip of the louse egg fixative from hair. Consequently, until now, and despite the availability of effective pediculicidal treatments, dealing with the eggshells persisting after an infestation has been an onerous task for most households. This type of product can enable families to deal more easily with persistent eggshells and improve self-esteem in affected children. creator: Elizabeth R. Brunton creator: Ian P. Whelan creator: Rebecca French creator: Mark N. Burgess creator: Ian F. Burgess uri: https://doi.org/10.7717/peerj.6759 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Brunton et al. title: Comparative characterization of biochars produced at three selected pyrolysis temperatures from common woody and herbaceous waste streams link: https://peerj.com/articles/6784 last-modified: 2019-04-15 description: Biochar, the product of biomass pyrolysis, has been explored as a soil amendment and carbon capture vessel. Recent literature has aligned biochar as a novel sorbent for a host of environmental contaminants. Through the variation of pyrolysis conditions, biochars can be engineered to have qualities desirable in sorbents whilst maintaining their agronomic benefits. This study focuses on identifying the effects that feedstock type and process temperature have on biochar characteristics which may in turn shed light on their potential environmental applications. Using this approach, six biochars were created from two waste biomasses. The biochars exhibited wide ranges of pH (5.6–11.1), surface area (16.2–397.4 m2/g), electrical conductivity (19–2,826 µS/cm), fixed carbon (72–97%), heavy metal and polycyclic aromatic hydrocarbons (PAHs). Statistically significant trends (P < 0.05) in biochar characteristics dependent upon increasing pyrolysis temperature and feedstock type were identified. Arsenic (>13 mg/kg), chromium (>93 mg/kg), copper (>143 mg/kg) and PAH (>6 mg/kg) concentrations presented themselves as obstacles to land application in a small number of biochars with respects to International Biochar Initiative (IBI) guidelines. However, it was demonstrated that these could be eliminated through employing pyrolysis processes which encompass higher temperatures (>500 °C) and ensuring the use of contaminant-free feedstocks. The variation in surface areas, carbonized fractions and surface functional groups achieved suggest that using the correct feedstock and process, biochar could be produced in Victoria (Australia) from common organic waste streams to the ends of acting as a sorbent, soil enhancer, and a waste management strategy. creator: Matthew Askeland creator: Bradley Clarke creator: Jorge Paz-Ferreiro uri: https://doi.org/10.7717/peerj.6784 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Askeland et al. title: Marine biodiversity research in the Ryukyu Islands, Japan: current status and trends link: https://peerj.com/articles/6532 last-modified: 2019-04-12 description: Marine biodiversity and derived ecosystem services are critical to the healthy functioning of marine ecosystems, and to human economic and societal well-being. Thus, an understanding of marine biodiversity in different ecosystems is necessary for their conservation and management. Coral reefs in particular are noted for their high levels of biodiversity, and among the world’s coral reefs, the subtropical Ryukyu Islands (RYS; also known as the Nansei Islands) in Japan have been shown to harbor very high levels of marine biodiversity. This study provides an overview of the state of marine biodiversity research in the RYS. First, we examined the amount of English language scientific literature in the Web of Science (WoS; 1995–2017) on six selected representative taxa spanning protists to vertebrates across six geographic sub-regions in the RYS. Our results show clear taxonomic and sub-region bias, with research on Pisces, Cnidaria, and Crustacea to be much more common than on Dinoflagellata, Echinodermata, and Mollusca. Such research was more commonly conducted in sub-regions with larger human populations (Okinawa, Yaeyama). Additional analyses with the Ocean Biogeographic Information System (OBIS) records show that within sub-regions, records are concentrated in areas directly around marine research stations and institutes (if present), further showing geographical bias within sub-regions. While not surprising, the results indicate a need to address ‘understudied’ taxa in ‘understudied sub-regions’ (Tokara, Miyako, Yakutane, Amami Oshima), particularly sub-regions away from marine research stations. Second, we compared the numbers of English language scientific papers on eight ecological topics for the RYS with numbers from selected major coral reef regions of the world; the Caribbean (CAR), Great Barrier Reef (GBR), and the Red Sea (RES). As expected, the numbers for all topics in the RYS were well below numbers from all other regions, yet within this disparity, research in the RYS on ‘marine protected areas’ and ‘herbivory’ was an order of magnitude lower than numbers in other regions. Additionally, while manuscript numbers on the RYS have increased from 1995 to 2016, the rate of increase (4.0 times) was seen to be lower than those in the CAR, RES, and GBR (4.6–8.4 times). Coral reefs in the RYS feature high levels of both endemism and anthropogenic threats, and subsequently they contain a concentration of some of the world’s most critically endangered marine species. To protect these threatened species and coral reef ecosystems, more data are needed to fill the research gaps identified in this study. creator: James Davis Reimer creator: Piera Biondi creator: Yee Wah Lau creator: Giovanni Diego Masucci creator: Xuan Hoa Nguyen creator: Maria E.A. Santos creator: Hin Boo Wee uri: https://doi.org/10.7717/peerj.6532 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Reimer et al. title: Do the regular annual extreme water level changes affect the seasonal appearance of Anabaena in Poyang Lake? link: https://peerj.com/articles/6608 last-modified: 2019-04-12 description: BackgroundPoyang Lake is an ecosystem experiencing annual variations in water level of up to 14 m. Water level changes were 8.03 and 11.22 m, respectively, in the years 2013 and 2014. The biomass and heterocyst frequency of Anabaena increased in the summers of recent years.MethodsA weekly to bi-weekly monitoring from June to November 2013 and 2014 was set up to explain the variations of Anabaena appearance in different phases of the water level.ResultsAnabaena was present in the lake throughout the year. The average relative biomass of Anabaena in the present study was over 40%, being most abundant in summer. The average heterocyst frequency was 0.23% in 2013 and 0.76% in 2014. Correlation analysis indicated a positive trend of Anabaena biomass with water temperature and water level and a negative one with total nitrogen (TN), which is the reason for the increase of heterocyst frequency in 2013 and 2014. Heterocyst frequency of Anabaena was positively correlated with water temperature, water level and PO4-P, and negatively with dissolved inorganic nitrogen (DIN/DIP), NO3-N and TN. Moreover, water temperature and DIN/DIP were significantly correlated with water level, indicating that water level changes have a direct effect on Anabaena and heterocyst formation in Poyang Lake.ConclusionsThe results of this study support the hypothesis that increasing biomass and heterocyst formation of Anabaena can be primarily caused by seasonal changes of the water level in Poyang Lake. creator: Kuimei Qian creator: Martin Dokulil creator: Yuwei Chen uri: https://doi.org/10.7717/peerj.6608 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Qian et al. title: Comparison of long-term kidney functions after radical nephrectomy and simple nephrectomy link: https://peerj.com/articles/6701 last-modified: 2019-04-12 description: ObjectiveTo determine if there is a difference in proceeding to CKD between patients who had undergone radical nephrectomy (RN) and simple nephrectomy (SN) for different indications by comparing the short- and long-term renal function.Materials and MethodsWe retrospectively analyzed the records of all patients who underwent nephrectomy (either for malign or benign indications) in our clinic between January 2007 and September 2017. The patients were divided into 2 groups according the type of surgery: 1) Radical nephrectomy Group, 2) Simple Nephrectomy Group. Renal function was evaluated with Glomerular Filtration Rate (GFR) calculated using the MDRD formula.ResultsA total of 276 patients were included in the study. There were 202 patients in RN Group and 74 patients in SN Group. The mean age of the patients in RN Group and SN Group were age 59,2 ± 11,5 and 49,9 ± 15,1 years, respectively (p = 0.001). GFR levels of patients in RN Group versus SN Group were as follows: Preoperative period: 84.9 vs. 81 mL/min/1.73 m2; postoperative 1st day: 60.5 vs. 84.4 mL/min/1.73 m2, postoperative 1st month 58.9 vs. 76 mL/min/1.73 m2, postoperative 1st year: 59.5 vs. 74.1 mL/min/1.73 m2; at last control 60.3 and 76.1 mL/min/1.73 m2. While preoperative GFR was found to be similar in two groups (p = 0.26), postoperative GFR values were found to be significantly lower in Group RN (p < 0.001). In comparison of the decrease in GFR in two groups at last follow-up, significantly higher decrease was observed in RN Group, 29% vs. 6%, (p < 0.05).ConclusionThe decrease in GFR exists more common and intensive after RN compared to SN. In long-term, compensation mechanisms that develop after sudden nephron loss like radical nephrectomy deteriorates kidney function more than gradual nephron loss as in benign etiologies which indicates simple nephrectomy. creator: Erkan Olcucuoglu creator: Senol Tonyali creator: Sedat Tastemur creator: Yusuf Kasap creator: Mehmet Emin Sirin creator: Eymen Gazel creator: Esin Olcucuoglu creator: Oner Odabas creator: Can Ates creator: Mahmut Taha Olcucu uri: https://doi.org/10.7717/peerj.6701 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Olcucuoglu et al. title: The prevalence of Pseudomonas aeruginosa and multidrug resistant Pseudomonas aeruginosa in healthy captive ophidian link: https://peerj.com/articles/6706 last-modified: 2019-04-12 description: BackgroundSnakes are globally considered as pet animals, and millions of ophidians are bred in captivity. Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can act as an opportunistic pathogen of man and animals and is frequently present in the oral and cloacal microbiota of healthy ophidians. It can cause severe clinical diseases and often shows antibiotic resistance. The aim of this study was to evaluate the prevalence and antibiotic resistance profiles of P. aeruginosa isolated from the cloacal microbiota of a large population sample of healthy captive ophidians and to evaluate the statistical associations with farming conditions.MethodsA total of 419 cloacal swabs were collected from snakes belonging to the Boidae (n = 45), Colubridae (n = 48) and Pythonidae (n = 326) families and inoculated onto complete culture media. Food, water and bedding samples were also analyzed. The antimicrobial susceptibility of P. aeruginosa isolates was evaluated through the Kirby-Bauer agar diffusion test. Statistical analyses were performed with the chi-square test.ResultsThe prevalence of P. aeruginosa was 59.9%, and 35.5% of these strains were multidrug resistant (MDR). The prevalence of MDR P. aeruginosa was significantly higher in adult samples than in young samples, and widespread resistance to Cephalosporins, Polymyxins and Sulfonamides was observed. Statistically significant differences in the prevalence of P. aeruginosa were observed depending on the farm size and snake family. Feeding thawed prey was associated with a higher P. aeruginosa and MDR P. aeruginosa prevalence. Moreover, snakes fed home-raised prey had a significantly higher MDR P. aeruginosa prevalence than snakes fed commercially available feed. Less frequent terrarium cleaning was associated with a higher MDR P. aeruginosa prevalence. On the other hand, snake reproductive status was not significantly associated with P. aeruginosa or MDR P. aeruginosa prevalence. All food, water and bedding samples were negative for P. aeruginosa presence.DiscussionThe overall P. aeruginosa prevalence found in this study was lower than that found by other authors, but a high proportion of the isolates were MDR. This study highlighted the presence of constitutive (such as age and taxonomic family) and managerial (farm size, cleaning cycle frequency and food type) factors associated with P. aeruginosa and/or MDR P. aeruginosa prevalence. Good breeding management and proper antibiotic treatment of P. aeruginosa infections could help reduce the presence of P. aeruginosa and MDR P. aeruginosa in the gut microbiota of snakes and consequently reduce the risk to public health. creator: Andrea Sala creator: Francesco Di Ianni creator: Igor Pelizzone creator: Mara Bertocchi creator: Davide Santospirito creator: Francesco Rogato creator: Sara Flisi creator: Costanza Spadini creator: Tiziano Iemmi creator: Emanuele Moggia creator: Enrico Parmigiani creator: Sandro Cavirani creator: Simone Taddei creator: Clotilde S. Cabassi uri: https://doi.org/10.7717/peerj.6706 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Sala et al. title: Identification and differential expression of piRNAs in the gonads of Amur sturgeon (Acipenser schrenckii) link: https://peerj.com/articles/6709 last-modified: 2019-04-12 description: ObjectiveSturgeons are considered living fossils, and have a very high conservation and economic value. Studies on the molecular mechanism of sturgeon gonadal development and sex differentiation would not only aid in understanding vertebrate sex determination but also benefit sturgeon aquaculture. Piwi-interacting RNAs (piRNAs) have been shown to function in germline or gonadal development. In this study, we performed small RNA deep sequencing and microarray hybridization to identify potential sturgeon piRNAs.MethodsMale and female sturgeon gonads were collected and used for small RNA sequencing on an Illumina HiSeq platform with the validation of piRNA expression by microarray chip. The program Bowtie and k-mer scheme were performed to filter small RNA reads and discover potential sturgeon piRNAs. A known piRNA database, the coding sequence (CDS), 5′ and 3′ untranslated region (UTR) database of the A. Schrenckii transcriptome, Gene Ontology (GO) database and KEGG pathway database were searched subsequently to analyze the potential bio-function of sturgeon piRNAs.ResultsA total of 875,679 putative sturgeon piRNAs were obtained, including 93 homologous to known piRNAs and hundreds showing sex-specific and sex-biased expression. Further analysis showed that they are predominant in both the ovaries and testes and those with a sex-specific expression pattern are nearly equally distribution between sexes. This may imply a relevant role in sturgeon gonadal development. KEGG pathway and GO annotation analyses indicated that they may be related to sturgeon reproductive processes.ConclusionOur study provides the first insights into the gonadal piRNAs in a sturgeon species and should serve as a useful resource for further elucidation of the gene regulation involved in the sex differentiation of vertebrates. These results should also facilitate the technological development of early sex identification in sturgeon aquaculture. creator: Lihong Yuan creator: Linmiao Li creator: Xiujuan Zhang creator: Haiying Jiang creator: Jinping Chen uri: https://doi.org/10.7717/peerj.6709 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Yuan et al. title: An experimental study on the early diagnosis of traumatic brain injury in rabbits based on a noncontact and portable system link: https://peerj.com/articles/6717 last-modified: 2019-04-12 description: Closed cerebral hemorrhage (CCH) is a common symptom in traumatic brain injury (TBI) patients who suffer intracranial hemorrhage with the dura mater remaining intact. The diagnosis of CCH patients prior to hospitalization and in the early stage of the disease can help patients get earlier treatments that improve outcomes. In this study, a noncontact, portable system for early TBI-induced CCH detection was constructed that measures the magnetic induction phase shift (MIPS), which is associated with the mean brain conductivity caused by the ratio between the liquid (blood/CSF and the intracranial tissues) change. To evaluate the performance of this system, a rabbit CCH model with two severity levels was established based on the horizontal biological impactor BIM-II, whose feasibility was verified by computed tomography images of three sections and three serial slices. There were two groups involved in the experiments (group 1 with 10 TBI rabbits were simulated by hammer hit with air pressure of 600 kPa by BIM-II and group 2 with 10 TBI rabbits were simulated with 650 kPa). The MIPS values of the two groups were obtained within 30 min before and after injury. In group 1, the MIPS values showed a constant downward trend with a minimum value of −11.17 ± 2.91° at the 30th min after 600 kPa impact by BIM-II. After the 650 kPa impact, the MIPS values in group 2 showed a constant downward trend until the 25th min, with a minimum value of −16.81 ± 2.10°. Unlike group 1, the MIPS values showed an upward trend after that point. Before the injury, the MIPS values in both group 1 and group 2 did not obviously change within the 30 min measurement. Using a support vector machine at the same time point after injury, the classification accuracy of the two types of severity was shown to be beyond 90%. Combined with CCH pathological mechanisms, this system can not only achieve the detection of early functional changes in CCH but can also distinguish different severities of CCH. creator: Jun Yang creator: Hui Zhao creator: Gen Li creator: Qisheng Ran creator: Jingbo Chen creator: Zelin Bai creator: Gui Jin creator: Jian Sun creator: Jia Xu creator: Mingxin Qin creator: Mingsheng Chen uri: https://doi.org/10.7717/peerj.6717 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Yang et al. title: Upstream sequence-dependent suppression and AtxA-dependent activation of protective antigens in Bacillus anthracis link: https://peerj.com/articles/6718 last-modified: 2019-04-12 description: The anthrax toxin is a virulence factor produced by the bacterium Bacillus anthracis. Transcription of anthrax toxin genes is controlled by the transcription factor AtxA. Thus, AtxA is thought to be a key factor for the pathogenicity of B. anthracis. Despite its important role in B. anthracis infection, the molecular mechanism by which AtxA controls expression of anthrax toxin remains unclear. This study aimed to characterize the molecular mechanism of AtxA-mediated regulation of protective antigen (PA), a component of anthrax toxin encoded by the pagA gene. First, the interaction between the upstream region of pagA and AtxA was evaluated in vivo by constructing a transcriptional fusion of the upstream region with an auxotrophic marker. The results showed that (i) the upstream region of pagA suppressed transcription of the downstream gene and (ii) AtxA recovered suppressed transcription. Second, in vitro analysis using a gel mobility shift assay was performed to evaluate binding specificity of the AtxA–DNA interaction. The result showed sequence-independent binding of AtxA to DNA. Taken together, our findings suggest that the expression of PA was suppressed by the upstream region of pagA and that an interaction of AtxA and the upstream region releases the suppression. creator: Kochi Toyomane creator: Yoshikazu Furuta creator: Daisuke Fujikura creator: Hideaki Higashi uri: https://doi.org/10.7717/peerj.6718 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Toyomane et al. title: Adaptive and degenerative evolution of the S-Phase Kinase-Associated Protein 1-Like family in Arabidopsis thaliana link: https://peerj.com/articles/6740 last-modified: 2019-04-12 description: Genome sequencing has uncovered tremendous sequence variation within and between species. In plants, in addition to large variations in genome size, a great deal of sequence polymorphism is also evident in several large multi-gene families, including those involved in the ubiquitin-26S proteasome protein degradation system. However, the biological function of this sequence variation is yet not clear. In this work, we explicitly demonstrated a single origin of retroposed Arabidopsis Skp1-Like (ASK) genes using an improved phylogenetic analysis. Taking advantage of the 1,001 genomes project, we here provide several lines of polymorphism evidence showing both adaptive and degenerative evolutionary processes in ASK genes. Yeast two-hybrid quantitative interaction assays further suggested that recent neutral changes in the ASK2 coding sequence weakened its interactions with some F-box proteins. The trend that highly polymorphic upstream regions of ASK1 yield high levels of expression implied negative expression regulation of ASK1 by an as-yet-unknown transcriptional suppression mechanism, which may contribute to the polymorphic roles of Skp1-CUL1-F-box complexes. Taken together, this study provides new evolutionary evidence to guide future functional genomic studies of SCF-mediated protein ubiquitylation. creator: Zhihua Hua creator: Zhenyu Gao uri: https://doi.org/10.7717/peerj.6740 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Hua and Gao title: Extracting the invisible: obtaining high quality DNA is a challenging task in small arthropods link: https://peerj.com/articles/6753 last-modified: 2019-04-12 description: BackgroundThe application of an appropriate extraction method is a relevant factor for the success of all molecular studies.MethodsSeven different DNA extraction methods suitable for high-throughput DNA sequencing with very small arthropods were compared by applying nine different protocols: three silica gel based spin methods, two cetyltrimethyl ammonium bromide (CTAB) based ones (one with an additional silica membrane), a protein precipitation method and a method based on a chelating resin (applying different protocols). The quantity (concentration) and quality (degradation, contamination, polymerase chain reaction (PCR) and sequencing success) of the extracted DNA as well as the costs, preparation times, user friendliness, and required supplies were compared across these methods. To assess the DNA quantity, two different DNA concentration measurements were applied. Additionally, the effect of varying amounts of starting material (different body sizes), variable lysis temperatures and mixing during DNA extraction was evaluated.ResultsAlthough low DNA concentrations were measured for all methods, the results showed that—with the exception of two methods—the PCR success was 100%. However, other parameters show vast differences. The time taken to perform DNA extraction varied from 20 min to 2.5 h (Chelex vs. CTAB) and the costs from 0.02 to 3.46 € (Chelex vs. QIAamp kit) per sample. High quality genomic DNA was only gained from four methods. Results of DNA quantity measurements further indicated that some devices cannot deal with small amounts of DNA and show variant results.DiscussionIn conclusion, using Chelex (chelating resin) turned out as a rapid, low-cost method which can provide high quality DNA for different kinds of molecular investigations. creator: Andrea Lienhard creator: Sylvia Schäffer uri: https://doi.org/10.7717/peerj.6753 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Lienhard and Schäffer title: Crowdsourcing in medical research: concepts and applications link: https://peerj.com/articles/6762 last-modified: 2019-04-12 description: Crowdsourcing shifts medical research from a closed environment to an open collaboration between the public and researchers. We define crowdsourcing as an approach to problem solving which involves an organization having a large group attempt to solve a problem or part of a problem, then sharing solutions. Crowdsourcing allows large groups of individuals to participate in medical research through innovation challenges, hackathons, and related activities. The purpose of this literature review is to examine the definition, concepts, and applications of crowdsourcing in medicine. This multi-disciplinary review defines crowdsourcing for medicine, identifies conceptual antecedents (collective intelligence and open source models), and explores implications of the approach. Several critiques of crowdsourcing are also examined. Although several crowdsourcing definitions exist, there are two essential elements: (1) having a large group of individuals, including those with skills and those without skills, propose potential solutions; (2) sharing solutions through implementation or open access materials. The public can be a central force in contributing to formative, pre-clinical, and clinical research. A growing evidence base suggests that crowdsourcing in medicine can result in high-quality outcomes, broad community engagement, and more open science. creator: Joseph D. Tucker creator: Suzanne Day creator: Weiming Tang creator: Barry Bayus uri: https://doi.org/10.7717/peerj.6762 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Tucker et al. title: Age, growth, mortality and recruitment of thin sharpbelly Toxabramis swinhonis Günther, 1873 in three shallow lakes along the middle and lower reaches of the Yangtze River basin, China link: https://peerj.com/articles/6772 last-modified: 2019-04-12 description: Despite being the most dominant and widespread small fish species in the lakes along the middle and lower reaches of the Yangtze River basin, Toxabramis swinhonis has been paid little attention by fisheries scientists and little is known about its population characteristics. For this reason, we estimated age, growth, mortality and recruitment of this species based on three shallow lakes, Biandantang Lake, Shengjin Lake and Kuilei Lake (BDT, SJH and KLH, respectively) in this region. A total of 13,585 (8,818 in BDT, 2,207 in SJH and 2,560 in KLH) individuals were collected during monthly sampling from July 2016 to September 2017. The results revealed that the age structures of T. swinhonis consisted of four age groups (0+–3+), with 0+–1+ year old fish comprising more than 98% of the samples. Allometric growth patterns were displayed by fish from all sampling sites and the von Bertalanffy growth functions estimated were Lt = 173.25 (1 – e−1.20 (t + 1.09)): BDT; Lt = 162.75 (1 – e−1.20 (t + 1.08)): SJH and Lt = 215.25 (1 – e−1.20 (t + 1.12)): KLH, respectively. The rates of total mortality (Z), natural mortality (M) and fishing mortality (F) at BDT, SJH and KLH were computed as 5.82, 5.50 and 4.55 year−1; 1.89, 1.87 and 1.75 year−1; 3.93, 3.63 and 2.80 year−1, respectively. Meanwhile, growth performance indices (φ′) were 0.68 (in BDT), 0.66 (in SJH) and 0.62 (in KLH), which indicated that T. swinhonis were overfished slightly in all study areas. Area-specific recruitment patterns were similar to each other, displaying evidence of batch spawning, with major peaks in April and August, accounting for 92.21% (BDT), 88.21% (SJH) and 88.73% (KLH) of total recruitment, respectively. These results showed that brief generation-time, fast growth rate, relatively high natural mortality rate and strong reproductive capacity (r-strategies) are reasons why this species became the most dominant species in many lakes of China. creator: Xianghong Dong creator: Tao Xiang creator: Tao Ju creator: Ruojing Li creator: Shaowen Ye creator: Sovan Lek creator: Jiashou Liu creator: Gaël Grenouillet uri: https://doi.org/10.7717/peerj.6772 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Dong et al. title: A small caseid synapsid, Arisierpeton simplex gen. et sp. nov., from the early Permian of Oklahoma, with a discussion of synapsid diversity at the classic Richards Spur locality link: https://peerj.com/articles/6615 last-modified: 2019-04-11 description: The fossil record of caseids, a clade of faunivorous to large herbivorous Permian synapsids, is unusual in having a poorly documented history. Although Kungurian caseids are common in the well-known continental deposits of North America, and the fossil record of the group extends into the middle Permian (Guadalupian), with the presence of the large caseid Ennatosaurus in the Mezen Basin faunal assemblage, only two other occurrences are known in older Permian age sediments. One is an undescribed caseid from the Bromacker Quarry in Germany, and the second is Oromycter from the lower Permian of Richards Spur, Oklahoma. The former is known from several articulated skeletons, but the latter is known only from a handful of skeletal elements, including elements of the snout and lower jaw, some phalanges, and a few vertebrae. Here the fragmentary tooth bearing elements and dorsal vertebrae of another small caseid from Richards Spur are described, with a discussion of its significance in the context of caseid evolution, and the continuously expanding faunal list and taxic diversity at this locality. creator: Robert R. Reisz uri: https://doi.org/10.7717/peerj.6615 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Reisz title: Variation in ant-mediated seed dispersal along elevation gradients link: https://peerj.com/articles/6686 last-modified: 2019-04-11 description: Ant-mediated seed dispersal, also known as myrmecochory, is a widespread and important mutualism that structures both plant and ant communities. However, the extent to which ant functional types (e.g., granivorous generalists vs. myrmecochorous ants) across environmental gradients affect seed removal rates is not fully understood. We used a replicated, standardized seed removal experiment along elevation gradients in four mountain ranges in the southwestern United States to test predictions that: (1) seed removal rates would be greater at lower elevations, and (2) seed species identity influences seed removal rates (i.e., seeds from their native elevation range would be removed at higher rates than seeds outside of their native elevation range). Both predictions were supported. Seed removal rates were ∼25% higher at lower elevation sites than at higher elevation sites. The low elevation Datura and high elevation Iris were removed at higher rates in their respective native ranges. We attribute observed differences in dispersal rates to changes in ant community composition, functional diversity, and abundance. We also suggest that temperature variation along the elevation gradient may explain these differences in seed removal rates. creator: Israel Del Toro creator: Relena R. Ribbons uri: https://doi.org/10.7717/peerj.6686 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Del Toro and Ribbons title: Preparation of mitochondria to measure superoxide flashes in angiosperm flowers link: https://peerj.com/articles/6708 last-modified: 2019-04-11 description: BackgroundMitochondria are the center of energy metabolism and the production of reactive oxygen species (ROS). ROS production results in a burst of “superoxide flashes”, which is always accompanied by depolarization of mitochondrial membrane potential. Superoxide flashes have only been studied in the model plant Arabidopsis thaliana using a complex method to isolate mitochondria. In this study, we present an efficient, easier method to isolate functional mitochondria from floral tissues to measure superoxide flashes.MethodWe used 0.5 g samples to isolate mitochondria within <1.5 h from flowers of two non-transgenic plants (Magnolia denudata and Nelumbo nucifera) to measure superoxide flashes. Superoxide flashes were visualized by the pH-insensitive indicator MitoSOX Red, while the mitochondrial membrane potential (ΔΨ m) was labelled with TMRM.ResultsMitochondria isolated using our method showed a high respiration ratio. Our results indicate that the location of ROS and mitochondria was in a good coincidence. Increased ROS together with a higher frequency of superoxide flashes was found in mitochondria isolated from the flower pistil. Furthermore, a higher rate of depolarization of the ΔΨ m was observed in the pistil. Taken together, these results demonstrate that the frequency of superoxide flashes is closely related to depolarization of the ΔΨ m in petals and pistils of flowers. creator: Chulan Zhang creator: Fengshuo Sun creator: Biao Xiong creator: Zhixiang Zhang uri: https://doi.org/10.7717/peerj.6708 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Zhang et al. title: Synopsis of the genus Ponthieva (Orchidaceae) in Colombia link: https://peerj.com/articles/6728 last-modified: 2019-04-11 description: The Neotropical orchid genus Ponthieva R. Br. was established in 1813. The 70 representatives of this taxon are terrestrial, lithophytic, or epiphytic plants with basal, sessile, or petiolate leaves which are glabrous or pubescent. Their erect peduncle is pubescent and terminated by lax, few- to many-flowered raceme. Flowers are non-resupinate, with dissimilar sepals and asymmetrical petals which are adnate to the sides of the gynostemium. The lip is uppermost, fused to the lower part of the gynostemium. Here the synopsis of Colombian Ponthieva is presented. A list of national genus representatives includes 26 species, including one new species, P. vallecaucana Szlach. Kolan. & Olędrzyńska, sp. nov., discovered during this study. We did not confirm occurrence of four species reported in previous research. To facilitate process of identification of genus representatives, we divided Ponthieva into six morphological complexes based on vegetative and floral characters. The highest number of species was found in Magdalena and Cauca Valley montane forests. Lectotypes for Ponthieva inaudita and P. mandonii are designated. Morphological characteristics and illustrations of perianth segments of Colombian representatives of the genus are provided as well as a key for their identification. creator: Dariusz L. Szlachetko creator: Marta Kolanowska creator: Natalia Olędrzyńska uri: https://doi.org/10.7717/peerj.6728 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Szlachetko et al. title: Respiratory syncytial virus, human metapneumovirus, and influenza virus infection in Bangkok, 2016-2017 link: https://peerj.com/articles/6748 last-modified: 2019-04-11 description: Children and adults residing in densely populated urban centers around the world are at risk of seasonal influenza-like illness caused by respiratory viruses such as influenza virus, human metapneumovirus (hMPV), and respiratory syncytial virus (RSV). In a large metropolitan of Thailand’s capital city Bangkok, most respiratory infections are rarely confirmed by molecular diagnostics. We therefore examined the frequency of RSV, hMPV, and influenza virus in 8,842 patients who presented influenza-like illness and sought medical care at a large hospital in Bangkok between 2016 and 2017. Using a multiplex real-time reverse-transcription polymerase chain reaction (RT-PCR), 30.5% (2,699/8,842) of nasopharyngeal (NP) swab samples tested positive for one or more of these viruses. Influenza virus comprised 17.3% (1,528/8,842), of which the majority were influenza A/H3N2. Such infection was most prevalent among adults and the elderly. RSV was identified in 11.4% (1,011/8,842) and were mostly ON1 and BA9 genotypes. Of the hMPV-positive samples (3.6%, 318/8,842), genotypes A2, B1, and B2 were detected. A small number of individuals experienced co-infections (1.8%, 155/8,842), most commonly between RSV and influenza A/H3N2. RSV and hMPV co-infections were also found, but mainly in young children. Viral respiratory tract infection peaked locally in the rainy season (June to September). These findings support the utility of rapid nucleic acid testing of RSV, hMPV, and influenza virus in patients with ILI. creator: Ilada Thongpan creator: Nungruthai Suntronwong creator: Preeyaporn Vichaiwattana creator: Nasamon Wanlapakorn creator: Sompong Vongpunsawad creator: Yong Poovorawan uri: https://doi.org/10.7717/peerj.6748 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Thongpan et al. title: Genome-wide analysis of maize OSCA family members and their involvement in drought stress link: https://peerj.com/articles/6765 last-modified: 2019-04-11 description: BackgroundWorldwide cultivation of maize is often impacted negatively by drought stress. Hyperosmolality-gated calcium-permeable channels (OSCA) have been characterized as osmosensors in Arabidopsis. However, the involvement of members of the maize OSCA (ZmOSCA) gene family in response to drought stress is unknown. It is furthermore unclear which ZmOSCA gene plays a major role in genetic improvement of drought tolerance in Maize.MethodsWe predicted the protein domain structure and transmembrane regions by using the NCBI Conserved Domain Database database and TMHMM server separately. The phylogeny tree was built by Mega7. We used the mixed linear model in TASSEL to perform the family-based association analysis.ResultsIn this report, 12 ZmOSCA genes were uncovered in the maize genome by a genome-wide survey and analyzed systematically to reveal their synteny and phylogenetic relationship with the genomes of rice, maize, and sorghum. These analyses indicated a relatively conserved evolutionary history of the ZmOSCA gene family. Protein domain and transmembrane analysis indicated that most of the 12 ZmOSCAs shared similar structures with their homologs. The result of differential expression analysis under drought at various stages, as well as the expression profiles in 15 tissues, revealed a functional divergence of ZmOSCA genes. Notably, the expression level of ZmOSCA4.1 being up-regulated in both seedlings and adult leaves. Notably, the association analysis between genetic variations in these genes and drought tolerance was detected. Significant associations between genetic variation in ZmOSCA4.1 and drought tolerance were found at the seedling stage. Our report provides a detailed analysis of the ZmOSCAs in the maize genome. These findings will contribute to future studies on the functional characterization of ZmOSCA proteins in response to water deficit stress, as well as understanding the mechanism of genetic variation in drought tolerance in maize. creator: Shuangcheng Ding creator: Xin Feng creator: Hewei Du creator: Hongwei Wang uri: https://doi.org/10.7717/peerj.6765 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Ding et al. title: Leaf anatomy and ultrastructure in senescing ancient tree, Platycladus orientalis L. (Cupressaceae) link: https://peerj.com/articles/6766 last-modified: 2019-04-11 description: Platycladus orientalis L. (Cupressaceae) has a lifespan of thousands of years. Ancient trees have very high scientific, economic and cultural values. The senescence of ancient trees is a new research area but is poorly understood. Leaves are the primary and the most sensitive organ of a tree. To understand leaf structural response to tree senescence in ancient trees, experiments investigating the morphology, anatomy and ultrastructure were conducted with one-year leaves of ancient P. orientalis (ancient tree >2,000 years) at three different tree senescent levels (healthy, sub-healthy and senescent) at the world’s largest planted pure forest in the Mausoleum of Yellow Emperor, Shaanxi Province, China. Observations showed that leaf structure significantly changed with the senescence of trees. The chloroplast, mitochondria, vacuole and cell wall of mesophyll cells were the most significant markers of cellular ultrastructure during tree senescence. Leaf ultrastructure clearly reflected the senescence degree of ancient trees, confirming the visual evaluation from above-ground parts of trees. Understanding the relationships between leaf structure and tree senescence can support decision makers in planning the protection of ancient trees more promptly and effectively by adopting the timely rejuvenation techniques before the whole tree irreversibly recesses. creator: Qianyi Zhou creator: Zhaohong Jiang creator: Xin Zhang creator: Tian Zhang creator: Hailan Zhu creator: Bei Cui creator: Yiming Li creator: Fei Zhao creator: Zhong Zhao uri: https://doi.org/10.7717/peerj.6766 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Zhou et al. title: Robust and accurate quantification of biomarkers of immune cells in lung cancer micro-environment using deep convolutional neural networks link: https://peerj.com/articles/6335 last-modified: 2019-04-10 description: Recent years have seen a growing awareness of the role the immune system plays in successful cancer treatment, especially in novel therapies like immunotherapy. The characterization of the immunological composition of tumors and their micro-environment is thus becoming a necessity. In this paper we introduce a deep learning-based immune cell detection and quantification method, which is based on supervised learning, i.e., the input data for training comprises labeled images. Our approach objectively deals with staining variation and staining artifacts in immunohistochemically stained lung cancer tissue and is as precise as humans. This is evidenced by the low cell count difference to humans of 0.033 cells on average. This method, which is based on convolutional neural networks, has the potential to provide a new quantitative basis for research on immunotherapy. creator: Lilija Aprupe creator: Geert Litjens creator: Titus J. Brinker creator: Jeroen van der Laak creator: Niels Grabe uri: https://doi.org/10.7717/peerj.6335 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Aprupe et al. title: Genetic data of museum specimens allow for inferring evolutionary history of the cosmopolitan genus Sirthenea (Heteroptera: Reduviidae) link: https://peerj.com/articles/6640 last-modified: 2019-04-10 description: Among the 30 known genera within subfamily Peiratinae, only the genus Sirthenea has a cosmopolitan distribution. The results of our studies are the first comprehensive analysis concerning one of the representatives of mentioned subfamily based on joint phylogenetic analyses of molecular and morphological data as well as molecular dating. A total of 32 species were included into the dataset with all known species of the genus Sirthenea. Material of over 400 dry specimens was examined for the morphological part of this study. The cosmopolitan distribution of Sirthenea and the inaccessibility of specimens preserved in alcohol required the extraction of DNA from the dried skeletal muscles of specimens deposited in 24 entomological collections. The oldest specimens used for the successful extraction and sequencing were collected more than 120 years ago in India. We performed Bayesian Inference analyses of molecular and morphological data separately, as well as combined analysis. The molecular and morphological data obtained during our research verify the correlation of the divergence dates of all known Sirthenea species. Results of the relaxed molecular clock analysis of the molecular data show that, the genus Sirthenea started diverging in the Late Cretaceous into two clades, which subsequently began to branch off in the Paleocene. Our results of phylogenetic analyses suggest that the fossula spongiosa and its development could be one of the most important morphological characters in the evolution of the genus, most likely associated with the ecological niche inhabited by Sirthenea representatives. Confirmation of the results obtained in our studies is the reconciliation of the evolutionary history of Sirthenea with the biogeographical processes that have shaped current global distribution of the genus. creator: Dominik Chłond creator: Natalia Sawka-Gądek creator: Dagmara Żyła uri: https://doi.org/10.7717/peerj.6640 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Chłond et al. title: Germination characteristics among different sheepgrass (Leymus chinensis) germplasm during the seed development and after-ripening stages link: https://peerj.com/articles/6688 last-modified: 2019-04-10 description: Sheepgrass (Leymus chinensis (Trin.) Tzvel) is an important forage grass in the Eurasian steppe. However, little information is available concerning its seed morphological features and germination characteristics during seed development and after-ripening among different germplasm. To clarify the appropriate seed harvest time and the effects of germplasm, seed development and after-ripening on seed germination, 20 germplasm of sheepgrass were selected. Moreover, the seed morphological and physical changes as well as the seed germination and dormancy characteristics of sheepgrass during seed development stages were analyzed using a seven—d gradient of day after pollination (DAP). The results indicated that the seed water content decreased significantly during 35–42 DAP and that the highest seed germination rate of most germplasm was observed at 35–42 DAP. Thus, 35–42 DAP may be the best time to harvest sheepgrass to obtain the maximum seed germination rate and avoid seed shattering. Furthermore, our results indicated that there were six types of germination patterns, including germplasm with increasing germination rates in the developing seed, such as S19 and S13, and germplasm that maintained a consistently low germination rate, such as S10. Moreover, we compared the seed germination rate of eight germplasm during seed development in both 2016 and 2017, and the results indicated that the seed germination patterns of the eight germplasm were highly consistent between the two consecutive years, suggesting that germplasm rather than year is the major factor in determining germination during seed development. The effect of after-ripening on seed germination was different among the germplasm where four types of germination patterns were revealed for 10 germplasm and resulted in various dormancy features. A two-factor ANOVA analysis suggested that the germplasm of the sheepgrass has a large influence on seed germination, whether during seed development or after-ripening. Thus, these findings lay the foundation for future studies on seed dormancy and germination and may guide the breeding of new cultivars of sheepgrass with better germination performance. creator: Weiguang Yang creator: Shu Liu creator: Guangxiao Yuan creator: Panpan Liu creator: Dongmei Qi creator: Xiaobing Dong creator: Hui Liu creator: Gongshe Liu creator: Xiaoxia Li uri: https://doi.org/10.7717/peerj.6688 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Yang et al. title: virMine: automated detection of viral sequences from complex metagenomic samples link: https://peerj.com/articles/6695 last-modified: 2019-04-10 description: Metagenomics has enabled sequencing of viral communities from a myriad of different environments. Viral metagenomic studies routinely uncover sequences with no recognizable homology to known coding regions or genomes. Nevertheless, complete viral genomes have been constructed directly from complex community metagenomes, often through tedious manual curation. To address this, we developed the software tool virMine to identify viral genomes from raw reads representative of viral or mixed (viral and bacterial) communities. virMine automates sequence read quality control, assembly, and annotation. Researchers can easily refine their search for a specific study system and/or feature(s) of interest. In contrast to other viral genome detection tools that often rely on the recognition of viral signature sequences, virMine is not restricted by the insufficient representation of viral diversity in public data repositories. Rather, viral genomes are identified through an iterative approach, first omitting non-viral sequences. Thus, both relatives of previously characterized viruses and novel species can be detected, including both eukaryotic viruses and bacteriophages. Here we present virMine and its analysis of synthetic communities as well as metagenomic data sets from three distinctly different environments: the gut microbiota, the urinary microbiota, and freshwater viromes. Several new viral genomes were identified and annotated, thus contributing to our understanding of viral genetic diversity in these three environments. creator: Andrea Garretto creator: Thomas Hatzopoulos creator: Catherine Putonti uri: https://doi.org/10.7717/peerj.6695 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Garretto et al. title: MetaMSD: meta analysis for mass spectrometry data link: https://peerj.com/articles/6699 last-modified: 2019-04-10 description: Mass spectrometry-based proteomics facilitate disease understanding by providing protein abundance information about disease progression. For the same type of disease studies, multiple mass spectrometry datasets may be generated. Integrating multiple mass spectrometry datasets can provide valuable information that a single dataset analysis cannot provide. In this article, we introduce a meta-analysis software, MetaMSD (Meta Analysis for Mass Spectrometry Data) that is specifically designed for mass spectrometry data. Using Stouffer’s or Pearson’s test, MetaMSD detects significantly more differential proteins than the analysis based on the single best experiment. We demonstrate the performance of MetaMSD using simulated data, urinary proteomic data of kidney transplant patients, and breast cancer proteomic data. Noting the common practice of performing a pilot study prior to a main study, this software will help proteomics researchers fully utilize the benefit of multiple studies (or datasets), thus optimizing biomarker discovery. MetaMSD is a command line tool that automatically outputs various graphs and differential proteins with confidence scores. It is implemented in R and is freely available for public use at https://github.com/soyoungryu/MetaMSD. The user manual and data are available at the site. The user manual is written in such a way that scientists who are not familiar with R software can use MetaMSD. creator: So Young Ryu creator: George A. Wendt uri: https://doi.org/10.7717/peerj.6699 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Ryu and Wendt title: Automated analysis of small RNA datasets with RAPID link: https://peerj.com/articles/6710 last-modified: 2019-04-10 description: Understanding the role of short-interfering RNA (siRNA) in diverse biological processes is of current interest and often approached through small RNA sequencing. However, analysis of these datasets is difficult due to the complexity of biological RNA processing pathways, which differ between species. Several properties like strand specificity, length distribution, and distribution of soft-clipped bases are few parameters known to guide researchers in understanding the role of siRNAs. We present RAPID, a generic eukaryotic siRNA analysis pipeline, which captures information inherent in the datasets and automatically produces numerous visualizations as user-friendly HTML reports, covering multiple categories required for siRNA analysis. RAPID also facilitates an automated comparison of multiple datasets, with one of the normalization techniques dedicated for siRNA knockdown analysis, and integrates differential expression analysis using DESeq2.Availability and ImplementationRAPID is available under MIT license at https://github.com/SchulzLab/RAPID. We recommend using it as a conda environment available from https://anaconda.org/bioconda/rapid creator: Sivarajan Karunanithi creator: Martin Simon creator: Marcel H. Schulz uri: https://doi.org/10.7717/peerj.6710 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Karunanithi et al. title: Decoupling species richness variation and spatial turnover in beta diversity across a fragmented landscape link: https://peerj.com/articles/6714 last-modified: 2019-04-10 description: BackgroundHow habitat fragmentation affects the relationship between local richness and the variation in community composition across space is important to both ecology and conservation biology, but this effect remains poorly understood.MethodsHere, we present an empirical study to address this topic in a fragmented landscape, the Thousand Island Lake (TIL), an artificial land-bridge island system with more than 1,000 islands, which provides an “experimental” fragmented landscape with a homogeneous matrix and similar successional history. We measured species composition and plant functional type (PFT) on 29 islands, and tested the effects of island area and isolation on the relationship between α- and β-diversity. General Linear Models were applied to test the impact of habitat fragmentation. In addition, variation partitioning was used to decouple α-diversity dependent and α-diversity independent spatial turnover in β-diversity of the plant community and across different PFTs.ResultsWe found habitat fragmentation influences β-diversity of plants primarily by modifying local α-diversity, not spatial turnover in the TIL system. We also found area-dependent environmental filtering and differential plant responses across functional types were the most likely underlying driving mechanisms.DiscussionThese results highlight the importance of hierarchical linkages between components of biodiversity across scales in fragmented landscapes, and have practical conservation implications. creator: Guang Hu creator: Maxwell C. Wilson creator: Jianguo Wu creator: Jingjing Yu creator: Mingjian Yu uri: https://doi.org/10.7717/peerj.6714 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Hu et al. title: The stimulating role of syringic acid, a plant secondary metabolite, in the microbial degradation of structurally-related herbicide, MCPA link: https://peerj.com/articles/6745 last-modified: 2019-04-10 description: The ability of microorganisms to degrade xenobiotics can be exploited to develop cost-effective and eco-friendly bioremediation technologies. Microorganisms can degrade almost all organic pollutants, but this process might be very slow in some cases. A promising way to enhance removal of recalcitrant xenobiotics from the environment lies in the interactions between plant exudates such as plant secondary metabolites (PSMs) and microorganisms. Although there is a considerable body of evidence that PSMs can alter the microbial community composition and stimulate the microbial degradation of xenobiotics, their mechanisms of action remain poorly understood. With this in mind, our aim was to demonstrate that similarity between the chemical structures of PSMs and xenobiotics results in higher micropollutant degradation rates, and the occurrence of corresponding bacterial degradative genes. To verify this, the present study analyses the influence of syringic acid, a plant secondary metabolite, on the bacterial degradation of an herbicide, 4-chloro-2-methylphenoxyacetic acid (MCPA). In particular, the presence of appropriate MCPA degradative genes, MCPA removal efficiency and changes in samples phytotoxicity have been analyzed. Significant MCPA depletion was achieved in samples enriched with syringic acid. The results confirmed not only greater MCPA removal from the samples upon spiking with syringic acid, and thus decreased phytotoxicity, but also the presence of a greater number of genes responsible for MCPA biodegradation. 16S rRNA gene sequence analysis revealed ubiquitous enrichment of the β-proteobacteria Rhodoferax, Achromobacter, Burkholderia and Cupriavidus. The obtained results provide further confirmation that plant metabolites released into the rhizosphere can stimulate biodegradation of xenobiotics, including MCPA. creator: Magdalena Urbaniak creator: Elżbieta Mierzejewska creator: Maciej Tankiewicz uri: https://doi.org/10.7717/peerj.6745 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Urbaniak et al. title: Pituitary tissue-specific miR-7a-5p regulates FSH expression in rat anterior adenohypophyseal cells link: https://peerj.com/articles/6458 last-modified: 2019-04-09 description: The follicle-stimulating hormone (FSH), which is synthesized and secreted by the anterior pituitary gland, plays an important role in regulating reproductive processes. In this study, using the TargetScan program, we predicted that microRNAs (miRNAs) regulate FSH secretion. Dual-luciferase reporter assays were performed and identified miR-7a-5p. MiR-7a-5p has been reported to regulate diverse cellular functions. However, it is unclear whether miR-7a-5p binds to mRNAs and regulates reproductive functions. Therefore, we constructed a suspension of rat anterior pituitary cells and cultured them under adaptive conditions, transfected miR-7a-5p mimics or inhibitor into the cell suspension and detected expression of the FSHb gene. The results demonstrated that miR-7a-5p downregulated FSHb expression levels, while treatment with miR-7a-5p inhibitors upregulated FSHb expression levels relative to those of negative control groups, as shown by quantitative PCR analysis. The results were confirmed with a subsequent experiment showing that FSH secretion was reduced after treatment with mimics and increased in the inhibitor groups, as shown by enzyme-linked immunosorbent assay. Our results indicated that miR-7a-5p downregulates FSHb expression levels, resulting in decreased FSH synthesis and secretion, which demonstrates the important role of miRNAs in the regulation of FSH and animal reproduction. creator: Chang-Jiang Wang creator: Hai-Xiang Guo creator: Dong-Xu Han creator: Ze-Wen Yu creator: Yi Zheng creator: Hao Jiang creator: Yan Gao creator: Bao Yuan creator: Jia-Bao Zhang uri: https://doi.org/10.7717/peerj.6458 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Wang et al. title: Transcriptional profiling in the livers of rats after hypobaric hypoxia exposure link: https://peerj.com/articles/6499 last-modified: 2019-04-09 description: Ascent to high altitude feels uncomfortable in part because of a decreased partial pressure of oxygen due to the decrease in barometric pressure. The molecular mechanisms causing injury in liver tissue after exposure to a hypoxic environment are widely unknown. The liver must physiologically and metabolically change to improve tolerance to altitude-induced hypoxia. Since the liver is the largest metabolic organ and regulates many physiological and metabolic processes, it plays an important part in high altitude adaptation. The cellular response to hypoxia results in changes in the gene expression profile. The present study explores these changes in a rat model. To comprehensively investigate the gene expression and physiological changes under hypobaric hypoxia, we used genome-wide transcription profiling. Little is known about the genome-wide transcriptional response to acute and chronic hypobaric hypoxia in the livers of rats. In this study, we carried out RNA-Sequencing (RNA-Seq) of liver tissue from rats in three groups, normal control rats (L), rats exposed to acute hypobaric hypoxia for 2 weeks (W2L) and rats chronically exposed to hypobaric hypoxia for 4 weeks (W4L), to explore the transcriptional profile of acute and chronic mountain sickness in a mammal under a controlled time-course. We identified 497 differentially expressed genes between the three groups. A principal component analysis revealed large differences between the acute and chronic hypobaric hypoxia groups compared with the control group. Several immune-related and metabolic pathways, such as cytokine-cytokine receptor interaction and galactose metabolism, were highly enriched in the KEGG pathway analysis. Similar results were found in the Gene Ontology analysis. Cogena analysis showed that the immune-related pathways were mainly upregulated and enriched in the acute hypobaric hypoxia group. creator: Zhenguo Xu creator: Zhilong Jia creator: Jinlong Shi creator: Zeyu Zhang creator: Xiaojian Gao creator: Qian Jia creator: Bohan Liu creator: Jixuan Liu creator: Chunlei Liu creator: Xiaojing Zhao creator: Kunlun He uri: https://doi.org/10.7717/peerj.6499 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Xu et al. title: Maturation trade-offs in octopus females and their progeny: energy, digestion and defence indicators link: https://peerj.com/articles/6618 last-modified: 2019-04-09 description: Sexual maturation and reproduction influence the status of a number of physiological processes and consequently the ecology and behaviour of cephalopods. Using Octopus mimus as a study model, the present work was focused in the changes in biochemical compound and activity that take place during gonadal maturation of females and its consequences in embryo and hatchlings characteristics. To do that, a total of 31 adult females of O. mimus were sampled to follow metabolites (ovaries and digestive gland) and digestive enzyme activities (alkaline and acidic proteases) during physiological and functional maturation. Levels of protein (Prot), triacylglyceride (TG), cholesterol (Chol), glucose (Glu), and glycogen (Gly) were evaluated. Groups of eggs coming from mature females were also sampled along development and after hatching (paralarvae of 1 and 3 days old) to track metabolites (Prot, TG, Glu, Gly, TG, Chol), digestive enzymes activity (Lipase, alkaline proteases, and acidic proteases), and antioxidant/detoxification defence indicators with embryos development. Based on the data obtained, we hypothesized that immature females store Chol in their ovaries, probably from the food they ingested, but switch to TG reserves at the beginning of the maturation processes. At the same time, results suggest that these processes were energetically supported by Glu, obtained probably from Gly breakdown by gluconeogenic pathways. Also, was observed that embryos metabolites and enzyme activities (digestive and antioxidant/detoxification enzymes) where maintained without significant changes and in a low activity during the whole organogenesis, meaning that organogenesis is relatively not energetically costly. In contrast, after organogenesis, a mobilization of nutrients and activation of the metabolic and digestive enzymes was observed, together with increments in consumption of yolk and Gly, and reduction in lipid peroxidation. Derived from our results, we also have the hypothesis that reactive oxygen species (ROS) were produced during the metabolic processes that occurs in ovarian maturation. Those ROS may be in part transferred to the egg provoking a ROS charge to the embryos. The elimination of ROS in embryos started when the activity of the heart and the absorption of the yolk around stages XIV and XV were evident. Altogether, these processes allowed the paralarvae to hatch with buffered levels of ROS and with the antioxidant defence mechanisms ready to support further ROS production derived from paralarvae higher life stage requirements (feeding and metabolic demands). creator: Alberto Olivares creator: Gabriela Rodríguez-Fuentes creator: Maite Mascaró creator: Ariadna Sanchez Arteaga creator: Karen Ortega creator: Claudia Caamal Monsreal creator: Nelly Tremblay creator: Carlos Rosas uri: https://doi.org/10.7717/peerj.6618 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Olivares et al. title: Evidence for an oncogenic role of HOXC6 in human non-small cell lung cancer link: https://peerj.com/articles/6629 last-modified: 2019-04-09 description: BackgroundIdentification of specific biomarkers is important for the diagnosis and treatment of non-small cell lung cancer (NSCLC). HOXC6 is a homeodomain-containing transcription factor that is highly expressed in several human cancers; however, its role in NSCLC remains unknown.MethodsThe expression and protein levels of HOXC6 were assessed in NSCLC tissue samples by Quantitative real-time PCR (qRT-PCR) and immunohistochemistry, respectively. HOXC6 was transfected into the NSCLC cell lines A549 and PC9, and used to investigate its effect on proliferation, migration, and invasion using CFSE, wound healing, and Matrigel invasion assays. Next-generation sequencing was also used to identify downstream targets of HOXC6 and to gain insights into the molecular mechanisms underlying its biological function.ResultsHOXC6 expression was significantly increased in 66.6% (20/30) of NSCLC tumor samples in comparison to normal controls. HOXC6 promoted proliferation, migration, and invasion of NSCLC cells in vitro. RNA-seq analysis demonstrated the upregulation of 310 and 112 genes in A549-HOXC6 and PC9-HOXC6 cells, respectively, and the downregulation of 665 and 385 genes in A549-HOXC6 and PC9-HOXC6 cells, respectively. HOXC6 was also found to regulate the expression of genes such as CEACAM6, SPARC, WNT6, CST1, MMP2, and KRT13, which have documented pro-tumorigenic functions.DiscussionHOXC6 is highly expressed in NSCLC, and it may enhance lung cancer progression by regulating the expression of pro-tumorigenic genes involved in proliferation, migration, and invasion. Our study highlighted the oncogenic potential of HOXC6, and suggests that it may be a novel biomarker for the diagnosis and treatment of NSCLC. creator: Yingcheng Yang creator: Xiaoping Tang creator: Xueqin Song creator: Li Tang creator: Yong Cao creator: Xu Liu creator: Xiaoyan Wang creator: Yan Li creator: Minglan Yu creator: Haisu Wan creator: Feng Chen uri: https://doi.org/10.7717/peerj.6629 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Yang et al. title: From climate zone to microhabitat—environmental factors affecting the coastal distribution of tiger beetles (Coleoptera: Cicindelidae) in the south-eastern European biodiversity hotspot link: https://peerj.com/articles/6676 last-modified: 2019-04-09 description: BackgroundTiger beetles (Coleoptera: Cicindelidae) are predatory insects usually occurring in various sandy habitats. In south-eastern Europe, especially in lowland areas located close to the sea coast, the diversity of Cicindelidae is one of the highest in the Palaearctic realm. Although previous studies conducted in different areas of the world show that many species are habitat specialists, unfortunately little is known about environmental factors affecting the diversity and distribution of tiger beetles in this region.Material and MethodsHabitat preferences for 12 tiger beetles taxa were analysed. Over 100 samples collected in eight countries located in coastal areas of the Black and Mediterranean Seas were studied, for which climate data, macrohabitat types, and soil parameters (soil humidity, salinity, pH, and structure) were investigated.ResultsMost studied Cicindelidae were characterised by narrow or very narrow habitat specialisation and did not co-occur with other ones, including 11 taxa found as habitat specialists occurring only in one or two types of macrohabitat. The most eurythopic species was Calomera littoralis nemoralis which occupied four macrohabitat types. The climatic zone, altitude, and humidity were found as the most important factors in the distribution of the studied tiger beetle species. Salt marshes and sandy sea beaches were noted as the most diverse macrohabitat types.DiscussionTiger beetle fauna of south-eastern Europe consists mainly of habitat specialists sensitive to environmental changes, which makes these beetles perfect bioindicators. Moreover, as a great number of studied Cicindelidae taxa occur in habitats which are under a significant human impact, we suggest that in the studied area the group can be successfully used as a flagship taxon for insect and nature conservation. creator: Radomir Jaskuła creator: Mateusz Płóciennik creator: Axel Schwerk uri: https://doi.org/10.7717/peerj.6676 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Jaskuła et al. title: Aridity thresholds of soil microbial metabolic indices along a 3,200 km transect across arid and semi-arid regions in Northern China link: https://peerj.com/articles/6712 last-modified: 2019-04-09 description: Soil microbial processes are crucial for understanding the ecological functions of arid and semi-arid lands which occupy approximately 40% of the global terrestrial ecosystems. However, how soil microbial metabolic activities may change across a wide aridity gradient in drylands remains unclear. Here, we investigated three soil microbial metabolic indices (soil organic carbon (SOC)-based microbial respiration, metabolic quotient, and microbial biomass as a proportion of total SOC) and the degree of carbon limitation for microbial respiration along a 3,200 km transect with a wide aridity gradient. The aridity gradient was customarily expressed using the aridity index (AI) which was calculated as the ratio of mean annual precipitation to mean annual evaporation, therefore, a lower AI value indicated a higher degree of aridity. Our results showed non-linear relationships between AI values and the metabolic indices with a clear aridity threshold for each of the three metabolic indices along the aridity gradient, respectively (AI = 0.13 for basal respiration, AI = 0.17 for metabolic quotient, and AI = 0.17 for MBC:SOC ratio). These metabolic indices linearly declined when AI was above the thresholds, but did not show any clear patterns when AI was below the thresholds. We also found that soil microbial respiration was highly limited by available carbon substrates at locations with higher primary production and relatively lower level of water limitation when AI was above the threshold, a counter-intuitive pattern that microbes were more starved in ecosystems with more substrate input. However, the increasing level of carbon limitation did correspond to the declining trend of the three metabolic indices along the AI gradient, which indicates that the carbon limitation influences microbial metabolism. We also found that the ratio of microbial biomass carbon to SOC in arid regions (AI < 0.2) with extremely low precipitation and primary production were not quantitatively related to SOC content. Overall, our results imply that microbial metabolism is distinctively different in arid lands than in non-arid lands. creator: Jianfeng Hou creator: Feike A. Dijkstra creator: Xiuwei Zhang creator: Chao Wang creator: Xiaotao Lü creator: Peng Wang creator: Xingguo Han creator: Weixin Cheng uri: https://doi.org/10.7717/peerj.6712 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Hou et al. title: Throwing light on dark diversity of vascular plants in China: predicting the distribution of dark and threatened species under global climate change link: https://peerj.com/articles/6731 last-modified: 2019-04-09 description: BackgroundAs global climate change accelerates, ecologists and conservationists are increasingly investigating changes in biodiversity and predicting species distribution based on species observed at sites, but rarely consider those plant species that could potentially inhabit but are absent from these areas (i.e., the dark diversity and its distribution). Here, we estimated the dark diversity of vascular plants in China and picked up threatened dark species from the result, and applied maximum entropy (MaxEnt) model to project current and future distributions of those dark species in their potential regions (those regions that have these dark species).MethodsWe used the Beals probability index to estimate dark diversity in China based on available species distribution information and explored which environmental variables had significant impacts on dark diversity by incorporating bioclimatic data into the random forest (RF) model. We collected occurrence data of threatened dark species (Eucommia ulmoides, Liriodendron chinense, Phoebe bournei, Fagus longipetiolata, Amentotaxus argotaenia, and Cathaya argyrophylla) and related bioclimatic information that can be used to predict their distributions. In addition, we used MaxEnt modeling to project their distributions in suitable areas under future (2050 and 2070) climate change scenarios.ResultsWe found that every study region’s dark diversity was lower than its observed species richness. In these areas, their numbers of dark species are ranging from 0 to 215, with a generally increasing trend from western regions to the east. RF results showed that temperature variables had a more significant effect on dark diversity than those associated with precipitation. The results of MaxEnt modeling showed that most threatened dark species were climatically suitable in their potential regions from current to 2070.DiscussionsThe results of this study provide the first ever dark diversity patterns concentrated in China, even though it was estimated at the provincial scale. A combination of dark diversity and MaxEnt modeling is an effective way to shed light on the species that make up the dark diversity, such as projecting the distribution of specific dark species under global climate change. Besides, the combination of dark diversity and species distribution models (SDMs) may also be of value for ex situ conservation, ecological restoration, and species invasion prevention in the future. creator: Lili Tang creator: Runxi Wang creator: Kate S. He creator: Cong Shi creator: Tong Yang creator: Yaping Huang creator: Pufan Zheng creator: Fuchen Shi uri: https://doi.org/10.7717/peerj.6731 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Tang et al. title: Salivary LDOC1 is a gender-difference biomarker of oral squamous cell carcinoma link: https://peerj.com/articles/6732 last-modified: 2019-04-09 description: BackgroundThe X-linked tumor suppressor gene LDOC1 is reported to be involved in oral cancer. The detection of biomarkers in salivary RNA is a non-invasive strategy for diagnosing many diseases. The aim of the present study was to investigate the potential of salivary LDOC1 as a biomarker of oral cancer.MethodsWe determined the expression levels of LDOC1 in the saliva of oral squamous cell carcinoma (OSCC) subjects, and investigated its correlation with various clinicopathological characteristics. The expression levels of salivary LDOC1 were detected in 53 OSCC subjects and 43 healthy controls using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. We used Fisher’s exact test to analyze the correlations between expression levels and clinicopathological characteristics.ResultsSalivary LDOC1 was significantly upregulated in females with OSCC (p = 0.0072), and significantly downregulated in males with OSCC (p = 0.0206). Eighty-nine percent of male OSCC subjects who smoked expressed low levels of LDOC1. OSCC cell lines derived from male OSCC subjects expressed low levels of LDOC1.ConclusionsA high level of salivary LDOC1 expression is a biomarker of OSCC in females. A high percentage of male OSCC subjects who smoke express low levels of salivary LDOC1. A low level of salivary LDOC1 expression is a biomarker of OSCC in males. creator: Chung-Ji Liu creator: Jen-Hao Chen creator: Shih-Min Hsia creator: Chiu-Chu Liao creator: Hui-Wen Chang creator: Tzong-Ming Shieh creator: Yin-Hwa Shih uri: https://doi.org/10.7717/peerj.6732 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Liu et al. title: Distribution patterns of dinoflagellate communities along the Songhua River link: https://peerj.com/articles/6733 last-modified: 2019-04-09 description: BackgroundDinoflagellates have the potential to pose severe ecological and economic damages to aquatic ecosystems. It is therefore largely needed to understand the causes and consequences of distribution patterns of dinoflagellate communities in order to manage potential environmental problems. However, a majority of studies have focused on marine ecosystems, while the geographical distribution patterns of dinoflagellate communities and associated determinants in freshwater ecosystems remain unexplored, particularly in running water ecosystems such as rivers and streams.MethodsHere we utilized multiple linear regression analysis and combined information on species composition recovered by high-throughput sequencing and spatial and environmental variables to analyze the distribution patterns of dinoflagellate communities along the Songhua River.ResultsAfter high-throughput sequencing, a total of 490 operational taxonomic units (OTUs) were assigned to dinoflagellates, covering seven orders, 13 families and 22 genera. Although the sample sites were grouped into three distinctive clusters with significant difference (p < 0.05) in environmental variables, OTUs-based dinoflagellate communities among the three clusters showed no significant difference (p > 0.05). Among all 24 environmental factors, two environmental variables, including NO3-N and total dissolved solids (TDS), were selected as the significantly influential factors (p < 0.05) on the distribution patterns of dinoflagellate communities based on forward selection. The redundancy analysis (RDA) model showed that only a small proportion of community variation (6.1%) could be explained by both environmental (NO3-N and TDS) and dispersal predictors (watercourse distance) along the River. Variance partitioning revealed a larger contribution of local environmental factors (5.85%) than dispersal (0.50%) to the total variation of dinoflagellate communities.DiscussionOur findings indicated that in addition to the two quantifiable processes in this study (species sorting and dispersal), more unquantifiable stochastic processes such as temporal extinction and colonization events due to rainfall may be responsible for the observed geographical distribution of the dinoflagellate community along the Songhua River. Results obtained in this study suggested that deeper investigations covering different seasons are needed to understand the causes and consequences of geographical distribution patterns of dinoflagellate biodiversity in river ecosystems. creator: Yangchun Gao creator: Yiyong Chen creator: Wei Xiong creator: Shiguo Li creator: Aibin Zhan uri: https://doi.org/10.7717/peerj.6733 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Gao et al. title: Temporal variability in zooplankton community in the western Yellow Sea and its possible links to green tides link: https://peerj.com/articles/6641 last-modified: 2019-04-08 description: Large-scale macro-algal blooms of Ulva prolifera (also called green tides) have appeared each summer since 2008 in the western Yellow Sea. In this study, we investigated the temporal variability in zooplankton community in the western Yellow Sea and its possible links to green tides using data from a long-term plankton survey off the coast of Qingdao, China. Environmental conditions observed in the study area during the green tide period (GTP: June–August, 2008–2013) were compared to the non-green tide period (NGTP: June–August, 2005–2007), to support the contention that variations observed in zooplankton community may be attributed to the green tides, as opposed to natural climatic or environmental variations. Zooplankton assemblage structure observed during the GTP was then compared to the NGTP. Significant variations were detected both in zooplankton abundance and assemblage structure between the two defined periods. The abundance of zooplankton, mainly copepods, was significantly decreased during the GTP. Meanwhile, the relative abundance of copepods decreased by approximately 10% and that of gelatinous zooplankton, including appendicularians, chaetognaths, and medusae, almost doubled (ca. increased by 6.4%). The dominant species of meroplankton completely changed, specifically, polychaeta, and echinoderm larvae were more dominant than decapod and bivalve larvae. With regard to zooplankton size structure, the NGTP showed a higher size diversity with more small-sized organisms, while the GTP showed a lower size diversity in the community. According to general linear models, the interannual variation in summer zooplankton abundance was significantly correlated with green tides. These results indicate that the temporal changes in zooplankton community may have a close link to the green tides. creator: Weicheng Wang creator: Guangtao Zhang creator: Xiaoxia Sun creator: Fang Zhang creator: Xing Zhang uri: https://doi.org/10.7717/peerj.6641 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Wang et al. title: Non-host class II ribonucleotide reductase in Thermus viruses: sequence adaptation and host interaction link: https://peerj.com/articles/6700 last-modified: 2019-04-08 description: Ribonucleotide reductases (RNR) are essential enzymes for all known life forms. Their current taxonomic distribution suggests extensive horizontal gene transfer e.g., by processes involving viruses. To improve our understanding of the underlying processes, we characterized a monomeric class II RNR (NrdJm) enzyme from a Thermus virus, a subclass not present in any sequenced Thermus spp. genome. Phylogenetic analysis revealed a distant origin of the nrdJm gene with the most closely related sequences found in mesophiles or moderate thermophiles from the Firmicutes phylum. GC-content, codon usage and the ratio of coding to non-coding substitutions (dN/dS) suggest extensive adaptation of the gene in the virus in terms of nucleotide composition and amino acid sequence. The NrdJm enzyme is a monomeric B12-dependent RNR with nucleoside triphosphate specificity. It exhibits a temperature optimum at 60–70 °C, which is in the range of the growth optimum of Thermus spp. Experiments in combination with the Thermus thermophilus thioredoxin system show that the enzyme is able to retrieve electrons from the host NADPH pool via host thioredoxin and thioredoxin reductases. This is different from other characterized viral RNRs such as T4 phage RNR, where a viral thioredoxin is present. We hence show that the monomeric class II RNR, present in Thermus viruses, was likely transferred from an organism phylogenetically distant from the one they were isolated from, and adapted to the new host in genetic signature and amino acids sequence. creator: Christoph Loderer creator: Karin Holmfeldt creator: Daniel Lundin uri: https://doi.org/10.7717/peerj.6700 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Loderer et al. title: Vegetation dynamics of abandoned paddy fields and surrounding wetlands in the lower Tumen River Basin, Northeast China link: https://peerj.com/articles/6704 last-modified: 2019-04-08 description: On the temperate lowland plain of the lower Tumen River, agricultural development has converted most marshland into paddy fields. However, the locations of old paddy fields in the lowland temperate zone, where the vegetation structure is dominated by herbs adapted to seasonally wet or waterlogged conditions, are poorly known, and the impact of land use history on marshland diversity and shifts in plant functional groups has been scantly researched. In this study, we used a chronosequence approach to investigate herbaceous wetland communities in different recovery phases (<5 years, 5–15 years, and >15 years), as well as natural wetland as a reference. We assessed their ecological characteristics, species composition and diversity to determine how they change during natural succession. Plant species composition and dominance in the abandoned fields changed markedly during natural secondary succession. Initially, the annual weeds Echinochloa crus-galli and Bidens tripartita were dominant. Later, communities gradually became dominated first by Polygonum thunbergii and then by tussock-forming Carex rostrata. Species diversity was higher in abandoned fields than in natural wetlands and decreased with time. The partition of β-diversity components revealed that replacement was the prominent process structuring plant communities in paddy field at different times since abandonment. Our results suggest that the vegetation of abandoned paddy fields could be restored effectively through natural succession, although there were some differences in plant functional group traits. Abandoned paddy fields may be good sites for restoration of wetland species and conservation of wetland habitat. creator: Guanglan Cao creator: Kazuaki Tsuchiya creator: Weihong Zhu creator: Toshiya Okuro uri: https://doi.org/10.7717/peerj.6704 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Cao et al. title: The unfolding of iRFP713 in a crowded milieu link: https://peerj.com/articles/6707 last-modified: 2019-04-08 description: The exploring of biological processes in vitro under conditions of macromolecular crowding is a way to achieve an understanding of how these processes occur in vivo. In this work, we study the unfolding of the fluorescent probe iRFP713 in crowded environment in vitro. Previously, we showed that the unfolding of the dimeric iRFP713 is accompanied by the formation of a compact monomer and an intermediate state of the protein. In the intermediate state, the macromolecules of iRFP713 have hydrophobic clusters exposed to the surface of the protein and are prone to aggregation. Concentrated solutions of polyethylene glycol (PEG-8000), Dextran-40 and Dextran-70 with a molecular mass of 8000, 40000 and 70000 Da, respectively, were used to model the conditions for macromolecular crowding. A limited available space provided by all the crowding agents used favors to the enhanced aggregation of iRFP713 in the intermediate state at the concentration of guanidine hydrochloride (GdnHCl), at which the charge of protein surface is neutralized by the guanidine cations. This is in line with the theory of the excluded volume. In concentrated solutions of the crowding agents (240–300 mg/ml), the stabilization of the structure of iRFP713 in the intermediate state is observed. PEG-8000 also enhances the stability of iRFP713 in the monomeric compact state, whereas in concentrated solutions of Dextran-40 and Dextran-70 the resistance of the protein in the monomeric state against GdnHCl-induced unfolding decreases. The obtained data argues for the excluded volume effect being not the only factor that contributes the behavior of biological molecules in a crowded milieu. Crowding agents do not affect the structure of the native dimer of iRFP713, which excludes the direct interactions between the target protein and the crowding agents. PEGs of different molecular mass and Dextran-40/Dextran-70 are known to influence the solvent properties of water. The solvent dipolarity/polarizability and basicity/acidity in aqueous solutions of these crowding agents vary in different ways. The change of the solvent properties in aqueous solutions of crowding agents might impact the functioning of a target protein. creator: Olesya V. Stepanenko creator: Olga V. Stepanenko creator: Irina M. Kuznetsova creator: Konstantin K. Turoverov uri: https://doi.org/10.7717/peerj.6707 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Stepanenko et al. title: Fluctuations in coral reef fish densities after environmental disturbances on the northern Great Barrier Reef link: https://peerj.com/articles/6720 last-modified: 2019-04-08 description: Global warming is predicted to increase the frequency and or severity of many disturbances including cyclones, storms, and prolonged heatwaves. The coral reef at Lizard Island, part of the Great Barrier Reef, has been recently exposed to a sequence of severe tropical cyclones (i.e., Ita in 2014 and Nathan in 2015) and a coral bleaching in the year 2016. Reef fishes are an essential part of the coral reef ecosystem, and their abundance is thus a good marker to estimate the magnitude of such disturbances. Here, we examined whether the recent disturbances at Lizard Island had an impact on the coral reef fish communities. To do this, we examined fish survey data collected before and after the disturbances for potential changes in total fish density post-disturbance. Also, by sorting fish species into 11 functional groups based on their trophic level (i.e., diet), we further explored the density changes within each functional group. Our findings showed an overall decline of 68% in fish density post-disturbance, with a significant density decrease in nine of 11 trophic groups. These nine groups were: browsers, corallivores, detritivores, excavator/scrapers, grazers, macro-invertivores, pisci-invertivores, planktivores, and spongivores. The piscivores, on the other hand, were the only “winners,” wherein their density showed an increase post-disturbance. These changes within functional groups might have a further impact on the trophodynamics of the food web. In summary, our findings provide evidence that the fish assemblage on the reefs around Lizard Island was considerably affected by extreme weather events, leading to changes in the functional composition of the reef fish assemblage. creator: Zegni Triki creator: Redouan Bshary uri: https://doi.org/10.7717/peerj.6720 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Triki and Bshary title: Ozymandias: a biodiversity knowledge graph link: https://peerj.com/articles/6739 last-modified: 2019-04-08 description: Enormous quantities of biodiversity data are being made available online, but much of this data remains isolated in silos. One approach to breaking these silos is to map local, often database-specific identifiers to shared global identifiers. This mapping can then be used to construct a knowledge graph, where entities such as taxa, publications, people, places, specimens, sequences, and institutions are all part of a single, shared knowledge space. Motivated by the 2018 GBIF Ebbe Nielsen Challenge I explore the feasibility of constructing a “biodiversity knowledge graph” for the Australian fauna. The data cleaning and reconciliation steps involved in constructing the knowledge graph are described in detail. Examples are given of its application to understanding changes in patterns of taxonomic publication over time. A web interface to the knowledge graph (called “Ozymandias”) is available at https://ozymandias-demo.herokuapp.com. creator: Roderic D.M. Page uri: https://doi.org/10.7717/peerj.6739 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Page title: Genotypic variation in disease susceptibility among cultured stocks of elkhorn and staghorn corals link: https://peerj.com/articles/6751 last-modified: 2019-04-08 description: Disease mortality has been a primary driver of population declines and the threatened status of the foundational Caribbean corals, Acropora palmata and A. cervicornis. There remain few tools to effectively manage coral disease. Substantial investment is flowing into in situ culture and population enhancement efforts, while disease takes a variable but sometimes high toll in restored populations. If genetic resistance to disease can be identified in these corals, it may be leveraged to improve resistance in restored populations and possibly lead to effective diagnostic tests and disease treatments. Using a standardized field protocol based on replicated direct-graft challenge assays, we quantified this important trait in cultured stocks from three field nurseries in the Florida Keys. Field tests of 12 genotypes of A. palmata and 31 genotypes of A. cervicornis revealed significant genotypic variation in disease susceptibility of both species measured both as risk of transmission (percent of exposed fragments that displayed tissue loss) and as the rate of tissue loss (cm2 d–1) in fragments with elicited lesions. These assay results provide a measure of relative disease resistance that can be incorporated, along with consideration of other important traits such as growth and reproductive success, into restoration strategies to yield more resilient populations. creator: Margaret W. Miller creator: Philip J. Colburn creator: Emma Pontes creator: Dana E. Williams creator: Allan J. Bright creator: Xaymara M. Serrano creator: Esther C. Peters uri: https://doi.org/10.7717/peerj.6751 license: http://creativecommons.org/publicdomain/zero/1.0/ rights: title: Efficacy and potential of phage therapy against multidrug resistant Shigella spp. link: https://peerj.com/articles/6225 last-modified: 2019-04-05 description: Shigella-infected bacillary dysentery or commonly known as Shigellosis is a leading cause of morbidity and mortality worldwide. The gradual emergence of multidrug resistant Shigella spp. has triggered the search for alternatives to conventional antibiotics. Phage therapy could be one such suitable alternative, given its proven long term safety profile as well as the rapid expansion of phage therapy research. To be successful, phage therapy will need an adequate regulatory framework, effective strategies, the proper selection of appropriate phages, early solutions to overcome phage therapy limitations, the implementation of safety protocols, and finally improved public awareness. To achieve all these criteria and successfully apply phage therapy against multidrug resistant shigellosis, a comprehensive study is required. In fact, a variety of phage-based approaches and products including single phages, phage cocktails, mutated phages, genetically engineered phages, and combinations of phages with antibiotics have already been carried out to test the applications of phage therapy against multidrug resistant Shigella. This review provides a broad survey of phage treatments from past to present, focusing on the history, applications, limitations and effective solutions related to, as well as the prospects for, the use of phage therapy against multidrug resistant Shigella spp. and other multidrug resistant bacterial pathogens. creator: Swee-Seong Tang creator: Sudhangshu Kumar Biswas creator: Wen Siang Tan creator: Ananda Kumar Saha creator: Bey-Fen Leo uri: https://doi.org/10.7717/peerj.6225 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Tang et al. title: Enhancement of carbon monoxide concentration in atmosphere due to large scale forest fire of Uttarakhand link: https://peerj.com/articles/6507 last-modified: 2019-04-05 description: The richly forested Indian state of Uttarakhand experienced widespread forest fires in April to May 2016. The current study examines dispersion of carbon monoxide (CO) from the source regions of forest fire to distant places, using the Lagrangian particle dispersion model, FLEXPART. Atmospheric Infrared Sounder (AIRS) observations revealed that CO columnar concentrations had increased by almost 28 percentage during 24 April to 02 May 2016 with respect to the previous non-burning period of April 2016 at Uttarakhand. It is also seen that there is considerable enhancement of 45 percentage in average columnar concentration of CO during the burning period, compared to that in the previous 5 years as observed by AIRS. In the present study, concentrations of CO at different pressure levels and columnar CO over Uttarakhand during the forest fire event have been simulated using FLEXPART. The area averaged profile of model derived CO has been compared with the profile from AIRS onboard Aqua. Comparison between model derived columnar CO and satellite observations shows good agreement with coefficient of correlation (r) approximately 0.91 over the burnt areas. Further analysis using FLEXPART reveals that the transport of pollutants is towards north-eastern and eastern regions from the locations of forest fire events. Model derived vertical distribution of CO over Tibet, which is situated at the north-east of Uttarakhand, shows significant increase of CO concentration at higher altitudes around 3 km from the mean sea level during the fire event. FLEXPART results show that the emissions from the Uttarakhand fires were transported to Tibet during the study period. creator: Jaya Thakur creator: Prajesh Thever creator: Biswadip Gharai creator: MVR Sesha Sai creator: VNRao Pamaraju uri: https://doi.org/10.7717/peerj.6507 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Thakur et al. title: Evaluation of potential molecular interaction between quorum sensing receptor, LuxP and grouper fatty acids: in-silico screening and simulation link: https://peerj.com/articles/6568 last-modified: 2019-04-05 description: Pathologically relevant behaviors of Vibrio, such as the expression of virulence factors, biofilm production, and swarming motility, have been shown to be controlled by quorum sensing. The autoinducer-2 quorum sensing receptor protein LuxP is one of the target proteins for drug development to suppress the virulence of Vibrio. Here, we reported the potential molecular interaction of fatty acids identified in vibriosis-resistant grouper with LuxP. Fatty acid, 4-oxodocosahexaenoic acid (4R8) showed significant binding affinity toward LuxP (−6.0 kcal/mol) based on molecular docking analysis. The dynamic behavior of the protein–ligand complex was illustrated by molecular dynamic simulations. The fluctuation of the protein backbone, the stability of ligand binding, and hydrogen bond interactions were assessed, suggesting 4R8 possesses potential interaction with LuxP, which was supported by the low binding free energy (−29.144 kJ/mol) calculated using the molecular mechanics Poisson–Boltzmann surface area. creator: Chen-Fei Low creator: Mohd Shahir Shamsir creator: Zeti-Azura Mohamed-Hussein creator: Syarul Nataqain Baharum uri: https://doi.org/10.7717/peerj.6568 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Low et al. title: Cafeteria diet increased adiposity in comparison to high fat diet in young male rats link: https://peerj.com/articles/6656 last-modified: 2019-04-05 description: BackgroundDietary intervention studies in animal models of obesity are crucial to elucidate the mechanistic effects of specific nutrients and diets. Although several models of diet induced obesity have been examined in rodents to assess obesity, there are few studies that have researched influence of different high fat and/or westernized diets. The aim of this study was to compare a high fat diet and a cafeteria diet on obesity related biochemical and physiological parameters in young male rats.MethodsFive week old Wistar male rats were fed a control chow diet (C), butter-based high fat diet (HF) or cafeteria diet (CAF) for twelve weeks. In HF, 40% of energy came from fat and this ratio was 46% in CAF. CAF composed of highly energetic and palatable human foods along with chow diet. At the end of the feeding protocol all animals were culled using CO2 asphyxia and cervical dislocation after an overnight fasting.ResultsTotal energy and fat intake of CAF was significantly higher than C and HF. CAF was more effective in inducing obesity, as demonstrated by increased weight gain, Lee index, fat depot weights and total body fat in comparison to C and HF. Despite increased adiposity in CAF, plasma glucose, insulin and HOMA-IR levels were similar between the groups. Plasma leptin and cholesterol levels were markedly higher in CAF than C and HF.DiscussionWe have demonstrated that there are differential effects of high fat diet and cafeteria diet upon obesity and obesity-related parameters, with CAF leading to a more pronounced adiposity in comparison to high fat diet in young male rats. Future studies should consider the varied outcomes of different diet induced obesity models and development of a standardized approach in similar research practices. creator: Yucel Buyukdere creator: Atila Gulec creator: Asli Akyol uri: https://doi.org/10.7717/peerj.6656 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Buyukdere et al. title: Properties of a cryptic lysyl oxidase from haloarchaeon Haloterrigena turkmenica link: https://peerj.com/articles/6691 last-modified: 2019-04-05 description: BackgroundLysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic.MethodsLOX open reading frame was cloned from Haloterrigena turkmenica in an E. coli expression vector. Recombinant Haloterrigena turkmenica lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting.ResultsCultured H. turkmenica has no detectable amine oxidase activity. HTU-LOX may be expressed in E. coli with a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu2+ at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein in H. turkmenica cells.ConclusionH. turkmenica contains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic.SignificanceThis is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa. creator: Nikolay B. Pestov creator: Daniel V. Kalinovsky creator: Tatyana D. Larionova creator: Alia Z. Zakirova creator: Nikolai N. Modyanov creator: Irina A. Okkelman creator: Tatyana V. Korneenko uri: https://doi.org/10.7717/peerj.6691 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Pestov et al. title: Diet and mitochondrial DNA haplotype of a sperm whale (Physeter macrocephalus) found dead off Jurong Island, Singapore link: https://peerj.com/articles/6705 last-modified: 2019-04-05 description: Despite numerous studies across the large geographic range of the sperm whale (Physeter macrocephalus), little is known about the diet and mitochondrial DNA haplotypes of this strongly female philopatric species in waters off Southeast Asia. A female sperm whale found dead in Singapore waters provided the opportunity to study her diet and mitochondrial DNA haplotype. Here we report on the identification of stomach contents and mitochondrial DNA haplotype of this individual, and we include coastal hydrodynamic modelling to determine the possible geographic origin of the whale. At least 28 species of prey were eaten by this adult female whale, most of which were cephalopods. The mesopelagic squids Taonius pavo, Histioteuthis pacifica, Chiroteuthis imperator,and Ancistrocheirus lesueurii made up over 65% of the whale’s stomach contents. Plastic debris was also found in the whale’s stomach. Based on the diet, genetics, and coastal hydrodynamic modelling that suggest an easterly drift of the whale carcass over several days, the dead sperm whale in Singapore probably originated from a pod in the Southern Indian Ocean. This study provides an increase in the understanding the diet and natural history of the sperm whale in Southeast Asia. The combined analyses of stomach contents, DNA, and hydrodynamic modeling could provide a context to future studies on the sperm whale strandings, and have broader applicability for other marine mammals in the region. creator: Marcus A.H. Chua creator: David J.W. Lane creator: Seng Keat Ooi creator: Serene H.X. Tay creator: Tsunemi Kubodera uri: https://doi.org/10.7717/peerj.6705 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Chua et al. title: HERVs, immunity, and autoimmunity: understanding the connection link: https://peerj.com/articles/6711 last-modified: 2019-04-05 description: Since their discovery in the 1960s, further investigation into endogenous retroviruses (ERVs) has challenged the conventional view of viral sequences as exclusively parasitic elements. Once presumed to be a group of passive genetic relics, it is becoming increasingly clear that this view of ERVs, while generally accurate, is incorrect in many specific cases. Research has identified ERV genes that appear to be co-opted by their mammalian hosts, but the biological function of ERV elements in humans remains a controversial subject. One area that has attracted some attention in this domain is the role of co-opted ERV elements in mammalian immune systems. The relationship between ERVs and human autoimmune diseases has also been investigated, but has historically been treated as a separate topic. This review will summarize the current evidence concerning the phenotypic significance of ERVs, both in the healthy immune system and in manifestations of autoimmunity. Furthermore, it will evaluate the relationship between these fields of study, and propose previously-unexplored molecular mechanisms through which human endogenous retroviruses might contribute to certain autoimmune pathologies. Investigation into these novel mechanisms could further our understanding of the molecular basis of autoimmune disease, and may one day provide new targets for treatment. creator: Matthew Greenig uri: https://doi.org/10.7717/peerj.6711 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Greenig title: Overexpression of lncRNA H19 changes basic characteristics and affects immune response of bovine mammary epithelial cells link: https://peerj.com/articles/6715 last-modified: 2019-04-05 description: The function of long non-coding RNA H19 (H19) on cell proliferation has been observed in various cell types, and the increased expression of H19 was also found in the lipopolysaccharide (LPS)-induced inflammatory bovine mammary epithelial cells (MAC-T). However, the roles of H19 in the inflammatory response and physiological functions of bovine mammary epithelial cell are not clear. In the present study, we found that overexpression of H19 in MAC-T cells significantly promoted cell proliferation, increased the protein and mRNA level of β-casein, and enhanced the expression of tight junction (TJ)-related proteins while inhibited staphylococcus aureus adhesion to cells. In addition, results demonstrated that overexpression of H19 affected the LPS-induced immune response of MAC-T cells by promoting expressions of inflammatory factors, including TNF-α, IL-6, CXCL2 and CCL5, and activating the NF-κB signal pathway. Our findings indicate that H19 is likely to play an important role in maintaining normal functions and regulating immune response of bovine mammary epithelial cells. creator: Xuezhong Li creator: Hao Wang creator: Yanfen Zhang creator: Jinjing Zhang creator: Shaopei Qi creator: Yong Zhang creator: Ming-Qing Gao uri: https://doi.org/10.7717/peerj.6715 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Li et al. title: Noninvasive and safe cell viability assay for Euglena gracilis using natural food pigment link: https://peerj.com/articles/6636 last-modified: 2019-04-04 description: Noninvasive and safe cell viability assay is required in many fields such as regenerative medicine, genetic engineering, single-cell analysis, and microbial food culture. In this case, a safe and inexpensive method which is a small load on cells and the environment is preferable without requiring expensive and space-consuming equipment and a technician to operate. We examined eight typical natural food pigments to find Monascus pigment (MP) or anthocyanin pigment (AP) works as a good viability indicator of dye exclusion test (DET) for Euglena gracilis which is an edible photosynthetic green microalga. This is the first report using natural food pigments as cell viability assay. Euglena gracilis stained by MP or AP can be visually judged with a bright field microscope. This was spectrally confirmed by scan-free, non-invasive absorbance spectral imaging A(x, y, λ) microscopy of single live cells and principal component analysis (PCA). To confirm the ability of staining dead cells and examine the load on the cells, these two natural pigments were compared with trypan blue (TB) and methylene blue (MP), which are synthetic dyes conventionally used for DET. As a result, MP and AP had as good ability of staining dead cells treated with microwave as TB and MB and showed faster and more uniform staining for dead cells in benzalkonium chloride than them. The growth curve and the ratio of dead cells in the culture showed that the synthetic dyes inhibit the growth of E. gracilis, but the natural pigments do not. As the cell density increased, however, AP increased the ratio of stained cells, which was prevented by the addition of glucose. MP can stain dead cells in a shorter time than AP, while AP is more stable in color against long-term irradiation of intense light than MP. Due to the low toxicity of these pigments, viability of cells in culture can be monitored with them over a long period. creator: Kyohei Yamashita creator: Koji Yamada creator: Kengo Suzuki creator: Eiji Tokunaga uri: https://doi.org/10.7717/peerj.6636 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Yamashita et al. title: Photo-hydrogen and lipid production from lactate, acetate, butyrate, and sugar manufacturing wastewater with an alternative nitrogen source by Rhodobacter sp. KKU-PS1 link: https://peerj.com/articles/6653 last-modified: 2019-04-04 description: Photo-hydrogen and lipid production from individual synthetic volatile fatty acids (VFAs) and sugar manufacturing wastewater (SMW) by Rhodobacter sp. KKU-PS1 with sodium glutamate or Aji-L (i.e., waste from the process of crystallizing monosodium glutamate) as a nitrogen source was investigated. Using individual synthetic VFAs, the maximum hydrogen production was achieved with Aji-L as a nitrogen source rather than sodium glutamate. The maximum hydrogen production was 1,727, 754 and 1,353 mL H2/L, respectively, using 25 mM of lactate, 40 mM of acetate and 15mM of butyrate as substrates. Under these conditions, lipid was produced in the range of 10.6–16.9% (w/w). Subsequently, photo-hydrogen and lipid production from SMW using Aji-L as nitrogen source was conducted. Maximal hydrogen production and hydrogen yields of 1,672 mL H2/L and 1.92 mol H2/mol substrate, respectively, were obtained. Additionally, lipid content and lipid production of 21.3% (w/w) and 475 mg lipid/L were achieved. The analysis of the lipid and fatty acid components revealed that triacyglycerol (TAG) and C18:1 methyl ester were the main lipid and fatty acid components, respectively, found in Rhodobacter sp. KKU-PS1 cells. creator: Thitirut Assawamongkholsiri creator: Alissara Reungsang creator: Sureewan Sittijunda uri: https://doi.org/10.7717/peerj.6653 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Assawamongkholsiri et al. title: Hematological parameters in largemouth bass (Micropterus salmoides) with formalin-preservation: comparison between wild tournament-caught and captive-raised fish link: https://peerj.com/articles/6669 last-modified: 2019-04-04 description: BackgroundLargemouth bass (Micropterus salmoides) are an economically important freshwater fish species that have been investigated for both the short and long-term effects of stress, secondary to angling. Limited data has been published on the hematological parameters of this species and blood sample stability is a notable limitation of hematologic field studies. A relatively novel technique using 10% neutral buffered formalin preserves heparinized whole blood and maintains blood cell stability beyond one month in striped bass. The objective of this study was to evaluate the differences in hematological parameters between tournament-caught and captive-raised largemouth bass using whole blood preservation with neutral buffered formalin.MethodsTwo populations of largemouth bass (n = 26 wild; n = 29 captive) underwent coccygeal venipuncture to collect heparinized whole blood for packed cell volume, total solids, and manual differential. Formalin preservation of heparinized whole blood facilitated manual hemocytometer analysis. Results were compared between the populations (tournament-caught, and captive-raised) with Wilcoxon rank sum test, a Hotelling’s T2 test, and Bonferroni simultaneous 95% confidence intervals to determine significance.ResultsThe mean packed cell volume (44.9 ± 5.4%) and total solids (7.2 ± 1.1 g/dL) were significantly higher, while the total leukocyte count (7.08 ± 1.86 × 103/µL) was significantly lower in the wild tournament-caught population of largemouth bass, as compared to the captive-raised counterparts (PCV 34.4 ± 7.2%; TS 5.2 ± 1.0 g/dL; WBC 16.43 ± 8.37 × 103/µL). The wild population demonstrated a significantly distinct leukogram characterized by a neutropenia (24.1 ± 12.7%), lymphocytosis (67.7 ± 13.0%), and monocytopenia (8.3 ± 2.9%), while the erythrocyte and thrombocyte counts were not significantly different between populations.DiscussionNumerous factors have been demonstrated to influence hematologic parameters in fish including age, size, sex, temperature, environmental oxygen level, population density, and infection. The wild population endured stress during angling capture, live-well hypoxia, transport, and extended air exposures at weigh in, which may have caused a stress leukopenia as well as osmoregulatory dysfunction and subsequent hemoconcentration. Further evaluation of seasonal impact as well as increased sample size is warranted to enhance our understanding of largemouth bass hematology.ConclusionThis study concluded that wild largemouth bass captured via tournament angling have higher packed cell volume and total solids, and lower total leukocyte counts, compared to captive-reared individuals. Through the completion of this study, we demonstrated the successful use of 10% neutral buffered formalin to preserve heparinized whole blood for precise hemocytometer cell counts in a new teleost species, the largemouth bass. creator: Michelle C. Whitehead creator: Chelsey L. Vanetten creator: Yaxin Zheng creator: Gregory A. Lewbart uri: https://doi.org/10.7717/peerj.6669 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Whitehead et al. title: Genome-wide characterization of the auxin response factor (ARF) gene family of litchi (Litchi chinensis Sonn.): phylogenetic analysis, miRNA regulation and expression changes during fruit abscission link: https://peerj.com/articles/6677 last-modified: 2019-04-04 description: Auxin response factors (ARFs) play fundamental roles in modulating various biological processes including fruit development and abscission via regulating the expression of auxin response genes. Currently, little is known about roles of ARFs in litchi (Litchi chinensis Sonn.), an economically important subtropical fruit tree whose production is suffering from fruit abscission. In this study, a genome-wide analysis of ARFs was conducted for litchi, 39 ARF genes (LcARFs) were identified. Conserved domain analysis showed that all the LcARFs identified have the signature B3 DNA-binding (B3) and ARF (Aux_rep) domains, with only 23 members having the dimerization domain (Aux_IAA). The number of exons in LcARF genes ranges from 2 to 16, suggesting a large variation for the gene structure of LcARFs. Phylogenetic analysis showed that the 39 LcARFs could be divided into three main groups: class I, II, and III. In total, 23 LcARFs were found to be potential targets of small RNAs, with three conserved and one novel miRNA-ARF (miRN43-ARF9) regulatory pathways discovered in litchi. Expression patterns were used to evaluate candidate LcARFs involved in various developmental processes, especially in flower formation and organ abscission. The results revealed that most ARF genes likely acted as repressors in litchi fruit abscission, that is, ARF2D/2E, 7A/7B, 9A/9B, 16A/16B, while a few LcARFs, such as LcARF5A/B, might be positively involved in this process. These findings provide useful information and resources for further studies on the roles of ARF genes in litchi growth and development, especially in the process of fruit abscission. creator: Yanqing Zhang creator: Zaohai Zeng creator: Chengjie Chen creator: Caiqin Li creator: Rui Xia creator: Jianguo Li uri: https://doi.org/10.7717/peerj.6677 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Zhang et al. title: Association and diagnostic value of serum SPINK4 in colorectal cancer link: https://peerj.com/articles/6679 last-modified: 2019-04-04 description: The role of serum serine peptidase inhibitor, Kazal type 4 (SPINK4), in colorectal cancer (CRC) is largely unknown. This study aimed to explore the association and diagnostic value of serum SPINK4 in CRC. A total of 70 preoperative CRC patients, 30 postoperative CRC patients, 30 gastric cancer patients, and 30 healthy controls were enrolled. Using enzyme-linked immunosorbent assays, we found that the serum SPINK4 level was significantly increased in preoperative CRC compared with postoperative CRC patients, gastric cancer patients, and healthy controls (p < 0.05). The serum SPINK4 level was remarkably elevated in colon cancer compared with rectal cancer and was enhanced in the M1 stage compared with the M0 stage (p < 0.05). The area under the receiver operating characteristic curve of serum SPINK4 level in the diagnosis of CRC was 0.9186, with a sensitivity and specificity of 0.886 and 0.900, respectively, and a cut-off value of 2.065. There was no significant difference between high and low expression of serum SPINK4 regarding the overall survival time and disease-free survival (p > 0.05). This study demonstrated that the serum SPINK4 level increased in CRC and was associated with the location and distant metastasis of CRC. It had a high diagnostic value in CRC but was not associated with the survival of CRC patients. creator: Mingzhi Xie creator: Kezhi Li creator: Jilin Li creator: Dongcheng Lu creator: Bangli Hu uri: https://doi.org/10.7717/peerj.6679 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Xie et al. title: Multi-gene phylogeny and divergence estimations for Evaniidae (Hymenoptera) link: https://peerj.com/articles/6689 last-modified: 2019-04-04 description: Ensign wasps (Hymenoptera: Evaniidae) develop as predators of cockroach eggs (Blattodea), have a wide distribution and exhibit numerous interesting biological phenomena. The taxonomy of this lineage has been the subject of several recent, intensive efforts, but the lineage lacked a robust phylogeny. In this paper we present a new phylogeny, based on increased taxonomic sampling and data from six molecular markers (mitochondrial 16S and COI, and nuclear markers 28S, RPS23, CAD, and AM2), the latter used for the first time in phylogenetic reconstruction. Our intent is to provide a robust phylogeny that will stabilize and facilitate revision of the higher-level classification. We also show the continued utility of molecular motifs, especially the presence of an intron in the RPS23 fragments of certain taxa, to diagnose evaniid clades and assist with taxonomic classification. Furthermore, we estimate divergence times among evaniid lineages for the first time, using multiple fossil calibrations. Evaniidae radiated primarily in the Early Cretaceous (134.1–141.1 Mya), with and most extant genera diverging near the K-T boundary. The estimated phylogeny reveals a more robust topology than previous efforts, with the recovery of more monophyletic taxa and better higher-level resolution. The results facilitate a change in ensign wasp taxonomy, with Parevania, and Papatuka, syn. nov. becoming junior synonyms of Zeuxevania, and Acanthinevania, syn. nov. being designated as junior synonym of Szepligetella. We transfer 30 species to Zeuxevania, either reestablishing past combinations or as new combinations. We also transfer 20 species from Acanthinevania to Szepligetella as new combinations. creator: Barbara J. Sharanowski creator: Leanne Peixoto creator: Anamaria Dal Molin creator: Andrew R. Deans uri: https://doi.org/10.7717/peerj.6689 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Sharanowski et al. title: Spiders did not repeatedly gain, but repeatedly lost, foraging webs link: https://peerj.com/articles/6703 last-modified: 2019-04-04 description: Much genomic-scale, especially transcriptomic, data on spider phylogeny has accumulated in the last few years. These data have recently been used to investigate the diverse architectures and the origin of spider webs, concluding that the ancestral spider spun no foraging web, that spider webs evolved de novo 10–14 times, and that the orb web evolved at least three times. These findings in fact result from a particular phylogenetic character coding strategy, specifically coding the absence of webs as logically equivalent, and homologous to, 10 other observable (i.e., not absent) web architectures. “Absence” of webs should be regarded as inapplicable data. To be analyzed properly by character optimization algorithms, it should be coded as “?” because these codes—or their equivalent—are handled differently by such algorithms. Additional problems include critical misspellings of taxon names from one analysis to the next (misspellings cause some optimization algorithms to drop terminals, which affects taxon sampling and results), and mistakes in spider natural history. In sum, the method causes character optimization algorithms to produce counter-intuitive results, and does not distinguish absence from secondary loss. Proper treatment of missing entries and corrected data instead imply that foraging webs are primitive for spiders and that webs have been lost ∼5–7 times, not gained 10–14 times. The orb web, specifically, may be homologous (originated only once) although lost 2–6 times. creator: Jonathan A. Coddington creator: Ingi Agnarsson creator: Chris A. Hamilton creator: Jason E. Bond uri: https://doi.org/10.7717/peerj.6703 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Coddington et al. title: Prehemodialysis arteriovenous access creation is associated with better cardiovascular outcomes in patients receiving hemodialysis: a population-based cohort study link: https://peerj.com/articles/6680 last-modified: 2019-04-03 description: BackgroundCardiovascular (CV) disease contributes to nearly half of the mortalities in patients with end-stage renal disease. Patients who received prehemodialysis arteriovenous access (pre-HD AVA) creation had divergent CV outcomes.MethodsWe conducted a population-based cohort study by recruiting incident patients receiving HD from 2001 to 2012 from the Taiwan National Health Insurance Research Database. Patients’ characteristics, comorbidities, and medicines were analyzed. The primary outcome of interest was major adverse cardiovascular events (MACEs), defined as hospitalization due to acute myocardial infarction, stroke, or congestive heart failure (CHF) occurring within the first year of HD. Secondary outcomes included MACE-related mortality and all-cause mortality in the same follow-up period.ResultsThe patients in the pre-HD AVA group were younger, had a lower burden of underlying diseases, were more likely to use erythropoiesis-stimulating agents but less likely to use renin–angiotensin–aldosterone system blockers. The patients with pre-HD AVA creation had a marginally lower rate of MACEs but a significant 35% lower rate of CHF hospitalization than those without creation (adjusted hazard ratio (HR) 0.65, 95% confidence interval (CI) [0.48–0.88]). In addition, the pre-HD AVA group exhibited an insignificantly lower rate of MACE-related mortality but a significantly 52% lower rate of all-cause mortality than the non-pre-HD AVA group (adjusted HR 0.48, 95% CI [0.39–0.59]). Sensitivity analyses obtained consistent results.ConclusionsPre-HD AVA creation is associated with a lower rate of CHF hospitalization and overall death in the first year of dialysis. creator: Cheng-Chieh Yen creator: Mei-Yin Liu creator: Po-Wei Chen creator: Peir-Haur Hung creator: Tse-Hsuan Su creator: Yueh-Han Hsu uri: https://doi.org/10.7717/peerj.6680 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Yen et al. title: A longitudinal study of the faecal microbiome and metabolome of periparturient mares link: https://peerj.com/articles/6687 last-modified: 2019-04-03 description: BackgroundPeriparturient mares are at increased risk of colic including large colon volvulus, which has a high mortality rate. Alterations in colonic microbiota related to either physiological or management changes, or both, that occur at this time have been suggested as potential causes for increased colic risk in this population of horses. Although the effect of management changes on the horse faecal microbiota has been investigated, limited work has been conducted to investigate changes in faecal microbiota structure and function in the periparturient period. The objectives of the current study were to investigate temporal stability of the faecal microbiota and volatile organic compounds (VOCs) of the faecal metabolome in periparturient mares.MethodsFaecal samples were collected weekly from five pregnant mares from 3 weeks pre-foaling to 7 weeks post-foaling. The microbiome data was generated by PCR amplification and sequencing of the V1–V2 regions of the bacterial 16S rRNA genes, while the VOC profile was characterised using headspace solid phase microextraction gas chromatography mass spectrometry.ResultsThe mare faecal microbiota was relatively stable over the periparturient period and most variation was associated with individual mares. A small number of operational taxonomic units were found to be significantly differentially abundant between samples collected before and after foaling. A total of 98 VOCs were identified. The total number of VOCs did not vary significantly between individual mares, weeks of sample collection and feeds available to the mares. Three VOCs (decane, 2-pentylfuran, and oct-2-ene) showed significant increase overtime on linear mixed effects modelling analysis. These results suggest that the mare faecal microbiota is structurally and functionally stable during the periparturient period. The findings also suggest that if changes in the gut microbiota are related to development of colic postpartum, altered risk may be due to inherent differences between individual mares. VOCs offer a cost-effective means of looking at the functional changes in the microbiome and warrant further investigation in mares at risk of colic. creator: Shebl E. Salem creator: Rachael Hough creator: Chris Probert creator: Thomas W. Maddox creator: Philipp Antczak creator: Julian M. Ketley creator: Nicola J. Williams creator: Sarah J. Stoneham creator: Debra C. Archer uri: https://doi.org/10.7717/peerj.6687 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Salem et al. title: Density of intertidal barnacles along their full elevational range of distribution conforms to the abundant-centre hypothesis link: https://peerj.com/articles/6719 last-modified: 2019-04-03 description: The abundant-centre hypothesis (ACH) predicts that the density of a species should peak at its distribution centre and decrease similarly towards distribution margins. The ACH has been deduced from a theory that postulates that environmental conditions should be most favourable for a species at the centre of its distribution. This idealised density pattern, however, has been supported by limited field studies, as natural patterns are often more complex. It is thus of interest to examine under what conditions compliance with the ACH could be favoured. Such conditions could be smooth environmental gradients with limited habitat patchiness throughout the distribution range of a species. Thus, we tested the ACH by measuring the density of an intertidal barnacle (Semibalanus balanoides) across its full vertical distribution range (from low to high elevations) on a rocky shore with similar substrate properties across elevations. To do a reliable test, we surveyed eight elevation zones applying an equal sampling effort in each zone. Average barnacle density conformed to the ACH, as it peaked at the middle of the vertical distribution range of this species. The same underlying theory predicts a similar unimodal pattern for maximum body size, but this trait was decoupled from density, as maximum barnacle size increased from low to high elevations. Overall, although the ACH is not a universal predictive tool as once envisioned, it may predict some cases well, as shown by this study. Therefore, the ACH should not be discarded completely, and its domain of application should be further evaluated. creator: Ricardo A. Scrosati creator: Matthew J. Freeman uri: https://doi.org/10.7717/peerj.6719 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Scrosati and Freeman title: Environmental modulation of the proteomic profiles from closely phylogenetically related populations of the red seaweed Plocamium brasiliense link: https://peerj.com/articles/6469 last-modified: 2019-04-02 description: The genus Plocamium encompasses seaweeds that are widely distributed throughout the world’s oceans, with Plocamium brasiliense found along the tropical and subtropical coasts of the Western Atlantic. This wide distribution can lead to structured populations due to environmental differences (e.g., light levels or temperature), restricted gene flow, and the presence of cryptic species. Abiotic variation can also affect gene expression, which consequently leads to differences in the seaweeds protein profile. This study aimed to analyze the genetic and proteomic profiles of P. brasiliense sampled in two geographically distinct sites on the coastline of Rio de Janeiro state, Brazil: Arraial do Cabo (P1) and Búzios (P2). The genetic profiles of macroalgal specimens from these two sites were indistinguishable as assessed by the markers UPA/23S, rbcL, and COI-5P; however, the protein profiles varied significantly between populations from the two sites. At both sites the ribulose-1,5-biphosphate carboxylase/oxygenase was the most abundant protein found in P. brasiliense specimens. The number of phycobiliproteins differed between both sites with the highest numbers being found at P1, possibly due to water depth. The differences in proteomic profiles of the two nearly identical populations of P. brasiliense suggest that environmental parameters such as light availability and desiccation might induce distinct protein expression, probably as a result of the phenotypic plasticity within this population of seaweed. creator: Gabriela Calegario creator: Lucas Freitas creator: Eidy Santos creator: Bruno Silva creator: Louisi Oliveira creator: Gizele Garcia creator: Cláudia Omachi creator: Renato Pereira creator: Cristiane Thompson creator: Fabiano Thompson uri: https://doi.org/10.7717/peerj.6469 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Calegario et al. title: Reproductive biology of little tunny Euthynnus alletteratus (Rafinesque, 1810) in the southwest Gulf of Mexico link: https://peerj.com/articles/6558 last-modified: 2019-04-02 description: The aim of this study was to describe the reproductive dynamic of Euthynnus alletteratus in the southwest Gulf of Mexico. The annual variation of the volume fraction occupied by gametes and tissues in gonads were related with main body indexes, such as the gonadosomatic index (IG), the hepatosomatic index (IH), and the nutrition index (IN), and compared with the sea surface temperature. A total of 951 E. alletteratus individuals were sampled, where a sex ratio of 1:1 and a size interval strongly skewed towards organisms with a fork length (LF) of 36–40 cm were observed. The IG showed an increase from March to September with maximum values in April and July. Two clearly defined peaks were observed, and they were consistent with the histological analysis, where the percentage of ripe gametes predominated from April to September. The optimum thermal window for reproductive activity was from 24 to 28 °C. The size of first sex maturity was 34.35 cm of LF for males and 34.60 (LF) for females, without significant difference between sexes. creator: Roberto Cruz-Castán creator: César Meiners-Mandujano creator: David Macías creator: Lourdes Jiménez-Badillo creator: Sergio Curiel-Ramírez uri: https://doi.org/10.7717/peerj.6558 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Cruz-Castán et al. title: Comparison of shallow-water seston among biogenic habitats on tidal flats link: https://peerj.com/articles/6616 last-modified: 2019-04-02 description: Aquatic structure-formers have the potential to establish mosaics of seston in shallow water if they modify the relative amounts of deposition (or filtration) and resuspension of particles. By sampling surface water adjacent to Lagrangian drifters traveling 0.1 to 2 m above the bottom, we tested the modification of seston in water masses flowing over two biogenic marine species (native eelgrass, Zostera marina; introduced oysters, Crassostrea gigas) in comparison to unstructured tidal flats. Water properties were examined at five intertidal sites in Washington State, USA, each with 27 drifts (three drifts at different stages of the tidal cycle in each of three patches of three habitat types; drift distance 116 m (109SD), duration 24 min (15SD)). At the initiation of each drift, habitat differences in water properties were already apparent: chlorophyll-a and total suspended solid (TSS) concentrations were greater in structured habitats than bare, and TSS was also inversely related to water depth. Water flowed more slowly across eelgrass than other habitat types. As water flowed across each habitat type, TSS generally increased, especially in shallow water, but without habitat differences; chlorophyll-a in these surface-water samples showed no consistent change during drifts. At higher TSS concentrations, quality in terms of organic content declined, and this relationship was not habitat-specific. However, quality in terms of chlorophyll-a concentration increased with TSS, as well as being greater in water over eelgrass than over other habitat types. These results support widespread mobilization of seston in shallow water ebbing or flooding across Washington State’s tidal flats, especially as water passes into patches of biogenic species. creator: Jennifer L. Ruesink creator: Cinde R. Donoghue creator: Micah J. Horwith creator: Alexander T. Lowe creator: Alan C. Trimble uri: https://doi.org/10.7717/peerj.6616 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Ruesink et al. title: Prevalence of malocclusion and occlusal traits in the early mixed dentition in Shanghai, China link: https://peerj.com/articles/6630 last-modified: 2019-04-02 description: BackgroundEpidemiological data on malocclusion among Chinese children are scant. The aim of this study was to provide detailed information on the prevalence of malocclusion in early mixed dentition children in Shanghai, China.MethodsA cross-sectional survey was conducted from September 2016 to April 2017, and 2,810 children aged 7- to 9- years were selected from 10 primary schools by cluster random sampling. Several occlusal parameters, including Angle molar relationship, overjet, overbite, open bite, anterior and posterior crossbite, midline displacement, scissors bite, and teeth crowding and spacing, were clinically registered by five calibrated orthodontic dentists.ResultsWe found that 79.4% children presented one or more occlusal anomalies. Angle Class I, Class II and Class III molar relationship were recorded in 42.3%, 50.9% and 5.9% of the sample, respectively. The proportion of Class III increased from 5.0% at age 7 to 7.8% at age 9. In the sagittal plane, increased overjet >3 mm was observed in 40.8% subjects, while the prevalence of severe overjet (>8 mm), anterior edge-to-edge (zero overjet) and anterior crossbite were 5.2%, 8.1% and 10.5%, respectively. Vertically, deep overbite >2/3 overlap was found in 6.2% of the children and open bite in 4.3%. Boys exhibited a higher rate of overbite than girls. For the transversal occlusal anomalies, 36.1% of the children had a midline displacement, which was followed by posterior crossbite (2.6%) and scissors bite (1.0%). Teeth space discrepancies were also common anomalies and anterior crowding (>2 mm) affecting 28.4% of the children, while anterior spacing (>4 mm) affecting 9.5%. Girls showed a higher prevalence of anterior crowding and a lower frequency of teeth spacing than boys.ConclusionsOur study demonstrated that malocclusion is prevalent among children in the early mixed dentition, and more health resources should be warranted to meet the challenge of prevention or early intervention of malocclusion. creator: Xin Yu creator: Hao Zhang creator: Liangyan Sun creator: Jie Pan creator: Yuehua Liu creator: Li Chen uri: https://doi.org/10.7717/peerj.6630 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Yu et al. title: On the half-life of thiocyanate in the plasma of the marine fish Amphiprion ocellaris: implications for cyanide detection link: https://peerj.com/articles/6644 last-modified: 2019-04-02 description: The illegal practice of using cyanide (CN) as a stunning agent to collect fish for both the marine aquarium and live fish food trades has been used throughout the Indo-Pacific for over 50 years. CN fishing is destructive to all life forms within the coral reef ecosystems where it is used and is certainly one of many anthropogenic activities that have led to 95% of the reefs in the Indo-Pacific being labeled at risk for degradation and loss. A field-deployable test for detecting fish caught using CN would assist in combating the use of this destructive practice, however, no reliable and robust test exists. Further, there is little toxicokinetic data available on marine fish to support the development of such a test, yet such data is critical to establishing the concentration range and time scale over which such a test would be viable. This study presents the first direct measurement of the half-life of the metabolite thiocyanate (SCN) after pulsed exposure to CN in a marine fish. SCN was measured in the plasma of Amphiprion ocellaris after exposure to 50 ppm CN for three exposure times (20, 45, and 60 s) using HPLC-UV and a C30 column pre-treated with polyethylene glycol. Plasma SCN levels observed are dose-dependent, reflecting a longer time for conversion of CN to SCN as the dose of CN increases. SCN plasma levels reached a maximum concentration (1.2–2.3 ppm) 12–20 h after exposure to CN. The half-life for the elimination of SCN was 1.01 ± 0.26 days for 45 s exposure and 0.44 ± 0.15 days for 20 s exposure. Fish were also directly exposed to SCN (100 ppm for 11 days) and the observed half-life for SCN elimination was 0.35 ± 0.07 days. Plasma SCN levels did not return to control levels, even after 41 days when exposed to CN but did return to control levels after 48 days when exposed to SCN. The similar half-lives observed for CN and SCN exposure suggests that SCN exposure can be used as a proxy for measuring the rate of SCN elimination following CN exposure. In order for plasma SCN to be used as a marker for CN exposure, these results must be extended to other species and endogenous levels of SCN in wild caught fish must be established. creator: Nancy E. Breen creator: J. Alexander Bonanno creator: Sara Hunt creator: Julia Grossman creator: Jordan Brown creator: Hannah Nolte creator: Andrew L. Rhyne uri: https://doi.org/10.7717/peerj.6644 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Breen et al. title: Revision of the Late Jurassic deep-water teleosauroid crocodylomorph Teleosaurus megarhinus Hulke, 1871 and evidence of pelagic adaptations in Teleosauroidea link: https://peerj.com/articles/6646 last-modified: 2019-04-02 description: Teleosauroids were a successful group of semi-aquatic crocodylomorphs that were an integral part of coastal marine/lagoonal faunas during the Jurassic. Their fossil record suggests that the group declined in diversity and abundance in deep water deposits during the Late Jurassic. One of the few known teleosauroid species from the deeper water horizons of the well-known Kimmeridge Clay Formation is ‘Teleosaurus’ megarhinus Hulke, 1871, a poorly studied, gracile longirostrine form. The holotype is an incomplete snout from the Aulacostephanus autissiodorensis Sub-Boreal ammonite Zone of Kimmeridge, England. The only other referred specimen is an almost complete skull from the slightly older A. eudoxus Sub-Boreal ammonite Zone of Quercy, France. Recently, the validity of this species has been called into question. Here we re-describe the holotype as well as the referred French specimen and another incomplete teleosauroid, DORCM G.05067i-v (an anterior rostrum with three osteoderms and an isolated tooth crown), from the same horizon and locality as the holotype. We demonstrate that all specimens are referable to ‘Teleosaurus’ megarhinus and that the species is indeed a valid taxon, which we assign to a new monotypic genus, Bathysuchus. In our phylogenetic analysis, the latest iteration of the ongoing Crocodylomorph SuperMatrix Project, Bathysuchus megarhinus is found as sister taxon to Aeolodon priscus within a subclade containing Mycterosuchus nasutus and Teleosaurus cadomensis. Notably Bathysuchus has an extreme reduction in dermatocranial ornamentation and osteoderm size, thickness and ornamentation. These features are mirrored in Aeolodon priscus, a species with a well-preserved post-cranial skeleton and a similar shallow and inconspicuous dermal ornamentation. Based on these morphological features, and sedimentological evidence, we hypothesise that the Bathysuchus + Aeolodon clade is the first known teleosauroid lineage that evolved a more pelagic lifestyle. creator: Davide Foffa creator: Michela M. Johnson creator: Mark T. Young creator: Lorna Steel creator: Stephen L. Brusatte uri: https://doi.org/10.7717/peerj.6646 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Foffa et al. title: Behavioral responses around conspecific corpses in adult eastern gorillas (Gorilla beringei spp.) link: https://peerj.com/articles/6655 last-modified: 2019-04-02 description: Humans were once considered unique in having a concept of death but a growing number of observations of animal responses to dying and dead conspecifics suggests otherwise. Complex arrays of behaviors have been described ranging from corpse removal and burial among social insects to quiet attendance and caregiving among elephants and primates. Less frequently described, however, are behavioral responses of individuals from different age/sex classes or social position toward the death of conspecifics. We describe behavioral responses of mountain gorillas (Gorilla beringei beringei) to the deaths of a dominant silverback and a dominant adult female from the same social group in Volcanoes National Park in Rwanda and the responses of Grauer’s gorillas (Gorilla b. graueri) to the corpse of an extra-group silverback in Kahuzi-Biega National Park, Democratic Republic of Congo. In gorillas, interactions between groups or with a lone silverback often result in avoidance or aggression. We predicted that: (i) more individuals should interact with the corpses of same-group members than with the corpse of the extra-group silverback; (ii) adult females with infants should avoid the corpse of the extra-group silverback; and (iii) in the mountain gorilla cases, individuals that shared close social relationships with the dead individual should spend more time with the corpse than other individuals in the group. We used a combination of detailed qualitative reports, photos, and videos to describe all occurrences of affiliative/investigative and agonistic behaviors observed at the corpses. We observed similar responses toward the corpses of group and extra-group individuals. Animals in all three cases showed a variety of affiliative/investigative and agonistic behaviors directed to the corpses. Animals of all age/sex classes interacted with the corpses in affiliative/investigative ways but there was a notable absence of all adult females at the corpse of the extra-group silverback. In all three cases, we observed only silverbacks and blackbacks being agonistic around and/or toward the corpses. In the mountain gorilla cases, the individuals who spent the most time with the corpses were animals who shared close social relationships with the deceased. We emphasize the similarity in the behavioral responses around the corpses of group and extra-group individuals, and suggest that the behavioral responses were influenced in part by close social relationships between the deceased and certain group members and by a general curiosity about death. We further discuss the implications close interactions with corpses have for disease transmission within and between gorilla social groups. creator: Amy Porter creator: Winnie Eckardt creator: Veronica Vecellio creator: Katerina Guschanski creator: Peter Philip Niehoff creator: Urbain Ngobobo-As-Ibungu creator: Radar Nishuli Pekeyake creator: Tara Stoinski creator: Damien Caillaud uri: https://doi.org/10.7717/peerj.6655 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Porter et al. title: Mapping-by-sequencing using NGS-based 3′-MACE-Seq reveals a new mutant allele of the essential nodulation gene Sym33 (IPD3) in pea (Pisum sativum L.) link: https://peerj.com/articles/6662 last-modified: 2019-04-02 description: Large collections of pea symbiotic mutants were accumulated in the 1990s, but the causal genes for a large portion of the mutations are still not identified due to the complexity of the task. We applied a Mapping-by-Sequencing approach including Bulk Segregant Analysis and Massive Analysis of cDNA Ends (MACE-Seq) sequencing technology for genetic mapping the Sym11 gene of pea which controls the formation of symbioses with both nodule bacteria and arbuscular-mycorrhizal fungi. For mapping we developed an F2-population from the cross between pea line N24 carrying the mutant allele of sym11 and the wild type NGB1238 (=JI0073) line. Sequencing libraries were prepared from bulks of 20 plants with mutant and 12 with wild-type phenotype. MACE-Seq differential gene expression analysis between mutant-phenotype and wild-type-phenotype bulks revealed 2,235 genes, of which 514 (23%) were up-regulated and 1,721 (77%) were down-regulated in plant roots inoculated with rhizobia as a consequence of sym11 mutation. MACE-Seq also detected single nucleotide variants between bulks in 217 pea genes. Using a novel mathematical model we calculated the recombination frequency (RF) between the Sym11 gene and these 217 polymorphic genes. Six genes with the lowest RF were converted into CAPS or dCAPS markers and genetically mapped on the complete mapping population of 108 F2-plants which confirmed their tight linkage to Sym11 and to each other. The Medicago truncatula Gaertn. (Mt) homologs of these genes are located in a distinct region of Mt chromosome 5, which corresponds to linkage group I of pea. Among 94 candidate genes from this region only one was down-regulated—the pea Sym33 homolog of the Mt IPD3 gene which is essential for nodulation. Sequencing of the Sym33 allele of the N24 (sym11) mutant revealed a single nucleotide deletion (c.C319del) in its third exon resulting in a codon shift in the open reading frame and premature translation termination. Thus, we identified a novel mutant allele sym33-4 most probably responsible for the mutant phenotype of the N24 (sym11) line, thereby demonstrating that mapping by MACE-Seq can be successfully used for genetic mapping of mutations and identification of candidate genes in pea. creator: Aleksandr I. Zhernakov creator: Oksana Y. Shtark creator: Olga A. Kulaeva creator: Jaroslava V. Fedorina creator: Alexey M. Afonin creator: Anna B. Kitaeva creator: Viktor E. Tsyganov creator: Fabian Afonso-Grunz creator: Klaus Hoffmeier creator: Björn Rotter creator: Peter Winter creator: Igor A. Tikhonovich creator: Vladimir A. Zhukov uri: https://doi.org/10.7717/peerj.6662 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Zhernakov et al. title: Spatial variation in allometric growth of invasive lionfish has management implications link: https://peerj.com/articles/6667 last-modified: 2019-04-02 description: Lionfish (Pterois volitans/miles) are an invasive species in the Western Atlantic and the Caribbean. Improving management of invasive lionfish populations requires accurate total biomass estimates, which depend on accurate estimates of allometric growth; sedentary species like lionfish often exhibit high levels of spatial variation in life history characteristics. We reviewed 17 published length-weight relationships for lionfish taken throughout their invasive range and found regional differences that led to significant misestimates when calculating weight from length observations. The spatial pattern we observed is consistent with findings from other studies focused on genetics or length-at-age. Here, the use of ex situ parameter values resulted in total biomass estimates between 76.2% and 140% of true observed biomass, and up to a threefold under- or overestimation of total weight for an individual organism. These findings can have implications for management in terms of predicting effects on local ecosystems, evaluating the effectiveness of removal programs, or estimating biomass available for harvest. creator: Juan Carlos Villaseñor-Derbez creator: Sean Fitzgerald uri: https://doi.org/10.7717/peerj.6667 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Villaseñor-Derbez and Fitzgerald title: Epizootic ulcerative syndrome causes cutaneous dysbacteriosis in hybrid snakehead (Channa maculata♀ × Channa argus♂) link: https://peerj.com/articles/6674 last-modified: 2019-04-02 description: Cutaneous microbiota play an important role in protecting fish against pathogens. Aphanomyces infection causes epizootic ulcerative syndrome (EUS) in fish, and by perturbing the integrity of the cutaneous microbiota, increases the potential for infection by pathogenic bacteria. However, whether the composition of the cutaneous microbiota is altered in fish with EUS, and if so, which species are changed and how this might influence infected fish, is still largely unclear. Considering the importance of cutaneous microbiota in maintaining host health, we hypothesized that Aphanomyces infection significantly enhances the presence of certain bacterial pathogens in the cutaneous microbiota and causes cutaneous dysbacteriosis. To test this hypothesis, we compared the cutaneous microbiota compositions of hybrid snakehead (Channa maculata♀ × Channa argus♂) with and without Aphanomyces infection using Illumina Miseq sequencing of the 16S rRNA gene. Our results showed that the cutaneous microbiota of hybrid snakehead were significantly altered subsequent to EUS infection and that the numbers of potentially pathogenic bacteria classified into the genera Anaerosinus, Anaerovorax, Dorea, and Clostridium were significantly enhanced in the cutaneous microbiota of hybrid snakehead with EUS, whereas bacteria classified into the genera Arthrobacter, Dysgonomonas, Anoxybacillus, Bacillus, Solibacillus, Carnobacterium, Lactococcus, Streptococcus, Achromobacter, Polynucleobacter, Vogesella, and Pseudomonas were significantly reduced. These results imply that treatment for EUS should not only take into consideration the control of Aphanomyces reproduction but should also focus on regulating the cutaneous microbiota of infected fish. creator: Zhifei Li creator: Guangjun Wang creator: Kai Zhang creator: Wangbao Gong creator: Ermeng Yu creator: Jingjing Tian creator: Jun Xie creator: Deguang Yu uri: https://doi.org/10.7717/peerj.6674 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Li et al. title: Mahalanobis distances and ecological niche modelling: correcting a chi-squared probability error link: https://peerj.com/articles/6678 last-modified: 2019-04-02 description: The Mahalanobis distance is a statistical technique that can be used to measure how distant a point is from the centre of a multivariate normal distribution. By measuring Mahalanobis distances in environmental space ecologists have also used the technique to model: ecological niches, habitat suitability, species distributions, and resource selection functions. Unfortunately, the original description of the Mahalanobis distance technique for ecological modelling contained an error describing how Mahalanobis distances could be converted into probabilities using a chi-squared distribution. This error has been repeated in the literature, and is present in popular modelling software. In the hope of correcting this error to maximise the potential application of the Mahalanobis distance technique within the ecological modelling community, I explain how Mahalanobis distances are calculated, and through a virtual ecology experiment demonstrate how to correctly produce probabilities and discuss the implications of the error for previous Mahalanobis distance studies. creator: Thomas R. Etherington uri: https://doi.org/10.7717/peerj.6678 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Etherington title: LncRNA MIR4435-2HG predicts poor prognosis in patients with colorectal cancer link: https://peerj.com/articles/6683 last-modified: 2019-04-02 description: BackgroundLncRNA MIR4435-2HG is observed in a variety of cancers, while its role in colorectal cancer is unknown. We aimed to demonstrate the relationship between MIR4435-2HG and colorectal cancer based on The Cancer Genome Atlas (TCGA) database.Materials and MethodsPatients with colorectal cancer were collected from TCGA. We compared the expression of MIR4435-2HG in colorectal cancer and normal tissues with Wilcoxon rank sum test, and logistic regression was used to evaluate the relationship between MIR4435-2HG and clinicopathological characters. Moreover, Kaplan–Meier and Cox regression was performed to evaluate the correlation between MIR4435-2HG and survival rate. Gene set enrichment analysis (GSEA) was also conducted to annotate biological function of MIR4435-2HG.ResultsMIR4435-2HG level was elevated in colorectal cancer tissues. Increased level of MIR4435-2HG was significantly correlated with TNM stage (OR = 1.66 for T1/T2 vs. T3/T4; OR = 1.68 for N0 vs. N1/N2), stage (OR = 1.66 for stage 1/2 vs. stage 3/4), and carcinoembryonic antigen level before treatment (OR = 1.70 for <5 vs. ≥5) (all P-value <0.05). High MIR4435-2HG expression had a poorer progression-free survival (p = 0.048), and overall survival (OS) (P = 0.028), which were validated in the GSE92921 and GSE29621 datasets. MIR4435-2HG expression (P = 0.040, HR = 1.955 (95% CI [1.031–3.710])) was independently correlated with OS. GSEA demonstrated that the P38/MAPK pathway, the VEGF pathway, the cell adhesion molecules cams, the NOD-like receptor signaling pathway, the cell surface interactions at the vascular wall, and integrin cell surface interactions were differentially enriched in MIR4435-2HG high expression phenotype.ConclusionsIncreased MIR4435-2HG might be a potential biomarker for the diagnosis and prognosis of colorectal cancer. Moreover, MIR4435-2HG might participate in the development of colorectal cancer via the P38/MAPK and VEGF pathway. creator: Wen Ouyang creator: Linlin Ren creator: Guohong Liu creator: Xiaosa Chi creator: Hongyun Wei uri: https://doi.org/10.7717/peerj.6683 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Ouyang et al. title: The cases for and against double-blind reviews link: https://peerj.com/articles/6702 last-modified: 2019-04-02 description: To date, the majority of authors on scientific publications have been men. While much of this gender bias can be explained by historic sexism and discrimination, there is concern that women may still be disadvantaged by the peer review process if reviewers’ biases lead them to reject publications with female authors more often. One potential solution to this perceived gender bias in the reviewing process is for journals to adopt double-blind reviews whereby neither the authors nor the reviewers are aware of each other’s identity and gender. To test the efficacy of double-blind reviews in one behavioral ecology journal (Behavioral Ecology, BE), we assigned gender to every authorship of every paper published for 2010–2018 in that journal compared to four other journals with single-blind reviews but similar subject matter and impact factors. While female authorships comprised only 35% of the total in all journals, the double-blind journal (BE) did not have more female authorships than its single-blind counterparts. Interestingly, the incidence of female authorship is higher at behavioral ecology journals (BE and Behavioral Ecology and Sociobiology) than in the ornithology journals (Auk, Condor, Ibis) for papers on all topics as well as those on birds. These analyses suggest that double-blind review does not currently increase the incidence of female authorship in the journals studied here. We conclude, at least for these journals, that double-blind review no longer benefits female authors and we discuss the pros and cons of the double-blind reviewing process based on our findings. creator: Amelia R. Cox creator: Robert Montgomerie uri: https://doi.org/10.7717/peerj.6702 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Cox and Montgomerie title: The Koala (Phascolarctos cinereus) faecal microbiome differs with diet in a wild population link: https://peerj.com/articles/6534 last-modified: 2019-04-01 description: BackgroundThe diet of the koala (Phascolarctos cinereus) is comprised almost exclusively of foliage from the genus Eucalyptus (family Myrtaceae). Eucalyptus produces a wide variety of potentially toxic plant secondary metabolites which have evolved as chemical defences against herbivory. The koala is classified as an obligate dietary specialist, and although dietary specialisation is rare in mammalian herbivores, it has been found elsewhere to promote a highly-conserved but low-diversity gut microbiome. The gut microbes of dietary specialists have been found sometimes to enhance tolerance of dietary PSMs, facilitating competition-free access to food. Although the koala and its gut microbes have evolved together to utilise a low nutrient, potentially toxic diet, their gut microbiome has not previously been assessed in conjunction with diet quality. Thus, linking the two may provide new insights in to the ability of the koala to extract nutrients and detoxify their potentially toxic diet.MethodThe 16S rRNA gene was used to characterise the composition and diversity of faecal bacterial communities from a wild koala population (n = 32) comprising individuals that predominately eat either one of two different food species, one the strongly preferred and relatively nutritious species Eucalyptus viminalis, the other comprising the less preferred and less digestible species Eucalyptus obliqua.ResultsAlpha diversity indices indicated consistently and significantly lower diversity and richness in koalas eating E. viminalis. Assessment of beta diversity using both weighted and unweighted UniFrac matrices indicated that diet was a strong driver of both microbial community structure, and of microbial presence/absence across the combined koala population and when assessed independently. Further, principal coordinates analysis based on both the weighted and unweighted UniFrac matrices for the combined and separated populations, also revealed a separation linked to diet. During our analysis of the OTU tables we also detected a strong association between microbial community composition and host diet. We found that the phyla Bacteroidetes and Firmicutes were co-dominant in all faecal microbiomes, with Cyanobacteria also co-dominant in some individuals; however, the E. viminalis diet produced communities dominated by the genera Parabacteroides and/or Bacteroides, whereas the E. obliqua-associated diets were dominated by unidentified genera from the family Ruminococcaceae.DiscussionWe show that diet differences, even those caused by differential consumption of the foliage of two species from the same plant genus, can profoundly affect the gut microbiome of a specialist folivorous mammal, even amongst individuals in the same population. We identify key microbiota associated with each diet type and predict functions within the microbial community based on 80 previously identified Parabacteroides and Ruminococcaceae genomes. creator: Kylie L. Brice creator: Pankaj Trivedi creator: Thomas C. Jeffries creator: Michaela D.J. Blyton creator: Christopher Mitchell creator: Brajesh K. Singh creator: Ben D. Moore uri: https://doi.org/10.7717/peerj.6534 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Brice et al. title: Influence of long term nitrogen limitation on lipid, protein and pigment production of Euglena gracilis in photoheterotrophic cultures link: https://peerj.com/articles/6624 last-modified: 2019-04-01 description: Nitrogen limitation is considered a good strategy for enhancement of algal lipid production while conversely N repletion has been shown to result in biomass rich in proteins. In this study, the influence of long-term N limitation on Euglena gracilis fatty acid (FA), protein, chlorophyll a, and carotenoid concentrations was studied in N limited cultures. Biomass composition was analyzed from three-time points from N starved late stationary phase cultures, exposed to three different initial N concentrations in the growth medium. Total lipid content increased under N limitation in ageing cultures, but the low N content and prolonged cultivation time resulted in the formation of a high proportion of saturated FAs. Furthermore, growth as well as the production of proteins, chlorophyll a and carotenoids were enhanced in higher N concentrations and metabolism of these cellular components stayed stable during the stationary growth phase. Our findings showed that a higher N availability and a shorter cultivation time is a good strategy for efficient E. gracilis biomass production, regardless of whether the produced biomass is intended for maximal recovery of polyunsaturated FAs, proteins, or photosynthetic pigments. Additionally, we showed an increase of neoxanthin, β-carotene, and diadinoxanthin as a response to higher N availability. creator: Marika Tossavainen creator: Usman Ilyass creator: Velimatti Ollilainen creator: Kalle Valkonen creator: Anne Ojala creator: Martin Romantschuk uri: https://doi.org/10.7717/peerj.6624 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Tossavainen et al. title: Obstetric and perinatal outcomes of dizygotic twin pregnancies resulting from in vitro fertilization versus spontaneous conception: a retrospective study link: https://peerj.com/articles/6638 last-modified: 2019-04-01 description: This study was designed to to assess perinatal and neonatal outcomes of dizygotic twin pregnancies conceived naturally or by in vitro fertilization (IVF). After strict selection, the study included 470 dizygotic twin pregnancies. There were 249 resulting from IVF treatments and 221 conceiving spontaneously. After adjusting maternal age and primiparity, the results showed that there were no significant differences between the two groups (P > 0.05) in terms of maternal antenatal complications and neonatal outcomes. In conclusion, our study does not reveal increased risks for pregnancy-related complications and adverse neonatal outcomes in dizygotic twin pregnancies following IVF treatments. With these fundamental data, this study could provide a reference for perinatal care and clinical assisted reproductive technology (ART) treatment and help to inform infertile parents about the potential risks of IVF treatments. creator: Hua Chen creator: Ying Wan creator: Haitao Xi creator: Weijue Su creator: Jing Cheng creator: Chunfang Zhu creator: Jieqiang Lv creator: Xinmei Wu creator: Junzhao Zhao uri: https://doi.org/10.7717/peerj.6638 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Chen et al. title: Complete plastome sequence of Iodes cirrhosa Turcz., the first in the Icacinaceae, comparative genomic analyses and possible split of Idoes species in response to climate changes link: https://peerj.com/articles/6663 last-modified: 2019-04-01 description: Plastome-based phylogenetic study has largely resolved the phylogeny of Icacinaceae. However, no single complete plastome sequence is available for Icacinaceae species, thereby limiting the further phylogenomics analysis of the members of this family. Here, we obtained the complete plastome sequence of Iodes cirrhosa Turcz., which is the first in Icacinaceae, by using the next-generation sequencing technology. The genome was annotated and compared with other closely related plastomes by using mVISTA. The divergence time of six Iodes species was analyzed using the BEAST software. The plastome of I. cirrhosa was 151,994 bp long, with a pair of inverted repeats (IRs, 24,973 bp) separated by a large single-copy (LSC, 84,527 bp) region and a small single-copy (SSC, 17,521 bp) region. The plastome encoded 112 unique genes, including 80 protein-coding, 28 tRNA, and four rRNA genes. Approximately 59 repeat sequences and 188 simple sequence repeats were identified. Four pairs of partially overlapped genes, namely, psbD/psbC, ndhF/Ψycf1, atpB/atpE, and rpl22/rps3, were observed. A comparison of the boundaries of the LSC, SSC, and IR regions with four other plastomes from Aquifoliales and Sapindales exhibited a high overall degree of sequence similarity. Four most highly variable regions, namely, trnH-GUG/psbA, psbM/trnD-GUC, petA/psbJ, and rps16/trnQ-UUG, were found. Using the plastome of I. cirrhosa as reference, we reassembled the plastomes of five Iodes species. Ka/Ks ratio analyses revealed that 27 genes and 52 amino acid residue sites from 11 genes had undergone strong positive selection in the Iodes branch, with the most abundant proteins being the NDH and ribosomal proteins. Divergence-time analysis indicated that Iodes species were first formed 34.40 million years ago. Results revealed that the ancestor of the six species was likely to have split in the late Eocene epoch. In summary, the first complete plastome sequence of I. cirrhosa provided valuable information regarding the evolutionary processes of Iodes species. creator: Liqiang Wang creator: Hui Zhang creator: Mei Jiang creator: Haimei Chen creator: Linfang Huang creator: Chang Liu uri: https://doi.org/10.7717/peerj.6663 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Wang et al. title: Microfluidic chip combined with magnetic-activated cell sorting technology for tumor antigen-independent sorting of circulating hepatocellular carcinoma cells link: https://peerj.com/articles/6681 last-modified: 2019-04-01 description: PurposeWe aimed to generate a capture platform that integrates a deterministic lateral displacement (DLD) microfluidic structure with magnetic-activated cell sorting (MACS) technology for miniaturized, efficient, tumor antigen-independent circulating tumor cell (CTC) separation.MethodsThe microfluidic structure was based on the theory of DLD and was designed to remove most red blood cells and platelets. Whole Blood CD45 MicroBeads and a MACS separator were then used to remove bead-labeled white blood cells. We established HepG2 human liver cancer cells overexpressing green fluorescent protein by lentiviral transfection to simulate CTCs in blood, and these cells were then used to determine the CTC isolation efficiency of the device. The performance and clinical value of our platform were evaluated by comparison with the Abnova CytoQuest™ CR system in the separating of blood samples from 12 hepatocellular carcinoma patients undergoing liver transplantation in a clinical follow-up experiment. The isolated cells were stained and analyzed by confocal laser scanning microscopy.ResultsUsing our integrated platform at the optimal flow rates for the specimen (60 µl/min) and buffer (100 µl/min per chip), we achieved an CTC yield of 85.1% ± 3.2%. In our follow-up of metastatic patients, CTCs that underwent epithelial–mesenchymal transition were found. These CTCs were missed by the CytoQuest™ CR bulk sorting approach, whereas our platform displayed increased sensitivity to EpCAMlow CTCs.ConclusionsOur platform, which integrates microfluidic and MACS technology, is an attractive method for high-efficiency CTC isolation regardless of surface epitopes. creator: Xuebin Wang creator: Liying Sun creator: Haiming Zhang creator: Lin Wei creator: Wei Qu creator: Zhigui Zeng creator: Ying Liu creator: Zhijun Zhu uri: https://doi.org/10.7717/peerj.6681 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Wang et al. title: Identification of the GRAS gene family in the Brassica juncea genome provides insight into its role in stem swelling in stem mustard link: https://peerj.com/articles/6682 last-modified: 2019-04-01 description: GRAS transcription factors are known to play important roles in plant signal transduction and development. A comprehensive study was conducted to explore the GRAS family in the Brassica juncea genome. A total of 88 GRAS genes were identified which were categorized into nine groups according to the phylogenetic analysis. Gene structure analysis showed a high group-specificity, which corroborated the gene grouping results. The chromosome distribution and sequence analysis suggested that gene duplication events are vital for the expansion of GRAS genes in the B. juncea genome. The changes in evolution rates and amino acid properties among groups might be responsible for their functional divergence. Interaction networks and cis-regulatory elements were analyzed including DELLA and eight interaction proteins (including four GID1, two SLY1, and two PIF3 proteins) that are primarily involved in light and hormone signaling. To understand their regulatory role in growth and development, the expression profiles of BjuGRASs and interaction genes were examined based on transcriptome data and qRT-PCR, and selected genes (BjuGRAS3, 5, 7, 8, 10, BjuB006276, BjuB037910, and BjuA021658) had distinct temporal expression patterns during stem swelling, indicating that they possessed diverse regulatory functions during the developmental process. These results contribute to our understanding on the GRAS gene family and provide the basis for further investigations on the evolution and functional characterization of GRAS genes. creator: Mengyao Li creator: Bo Sun creator: Fangjie Xie creator: Ronggao Gong creator: Ya Luo creator: Fen Zhang creator: Zesheng Yan creator: Haoru Tang uri: https://doi.org/10.7717/peerj.6682 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Li et al. title: Eyes in Staurozoa (Cnidaria): a review link: https://peerj.com/articles/6693 last-modified: 2019-04-01 description: The presence of dark pigment spots associated with primary tentacles (or structures derived from them, i.e., rhopalioids) in Staurozoa was recently overlooked in a study on the evolution of cnidarian eyes (defined as a “region made of photoreceptor cells adjacent to pigment cells”, irrespective of image formation, i.e., including all photoreceptive organs). Review of old and recent literature on Staurozoa shows that dark pigment spots are present in virtually all species of Manania, as well as some species of Haliclystus, Stylocoronella, and probably Calvadosia. The known ultrastructure of ocelli seems to be compatible with light perception, but no immediate response to changes in light intensity have been observed in the behavior of staurozoans. Therefore, although further studies addressing photic behavior are required, we discuss an earlier hypothesis that the dark spots in some stauromedusae may be related to synchronous spawning, as well as the possible sensorial function of rhopalioids. Observations summarized here suggest a possible ninth independent origin of eyes in Cnidaria, within a lineage of benthic medusae. Alternatively, documented similarity across medusae of Cubozoa, Scyphozoa, and Staurozoa—with eyes being topologically associated with primary tentacles in each of these taxa—could indicate shared ancestry and a single origin of eyes in this clade known as Acraspeda. Information on Staurozoa, one of the least studied groups within Cnidaria, is often neglected in the literature, but correctly recognizing the characters of this class is crucial for understanding cnidarian evolution. creator: Lucília Souza Miranda creator: Allen Gilbert Collins uri: https://doi.org/10.7717/peerj.6693 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2019 Miranda and Collins title: Rapid screening mutations of first-line-drug-resistant genes in Mycobacterium tuberculosis strains by allele-specific real-time quantitative PCR link: https://peerj.com/articles/6696 last-modified: 2019-04-01 description: Tuberculosis (TB) is a worldwide health, economic, and social burden, especially in developing countries. Drug-resistant TB is the most serious type of this burden. Thus, it is necessary to screen drug-resistant mutations by using a simple and rapid detection method. A total of 32 pairs of allele-specific PCR (AS-PCR) primers were designed to screen mutation and/or wild-type alleles of 16 variations in four first-line drug-resistant genes (katG, rpoB, rpsL, and embB) of TB strains. A pair of primers was designed to amplify 16S rRNA gene and to verify successful amplification. Subsequently, we tested the specificity and sensitivity of these AS-PCR primers. The optimized condition of these AS-PCR primers was first confirmed. All mutations could be screened in general AS-PCR, but only 13 of 16 variations were intuitively investigated by using real-time quantitative PCR (qPCR) and AS-PCR primers. The results of specificity assay suggested that the AS-PCR primers with mutation and/or wildtype alleles could successfully amplify the corresponding allele under optimized PCR conditions. The sensitivity of nine pairs of primers was 500 copy numbers, and the other seven pairs of primers could successfully amplify correct fragments with a template comprising 103 or 104 copy numbers template. An optimized AS-qPCR was established to screen drug-resistant mutations in TB strains with high specificity and sensitivity. creator: Pengpeng Yang creator: Yuzhu Song creator: Xueshan Xia creator: A-Mei Zhang uri: https://doi.org/10.7717/peerj.6696 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2019 Yang et al.