title: PeerJ description: Articles published in PeerJ link: https://peerj.com/articles/index.rss3?journal=peerj&page=1397 creator: info@peerj.com PeerJ errorsTo: info@peerj.com PeerJ language: en 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.