Efficient generation of human primordial germ cell-like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale
Background The mechanisms underlying human germ cell development and infertility remain largely unknown due to bioethical issues and the shortage of experimental materials. Therefore, an effective in vitro induction system of human primordial germ-like cells (hPGCLCs) from human pluripotent stem cells (hPSC) is in high demand. The current strategies used for the generation of hPGCLCs are not only costly but also difficult to perform at a large scale, thereby posing barriers to further research. In this study, we attempted to solve these problems by providing a new 3D culture system for hPGCLC differentiation. Methods The efficiency and relative yield of a methylcellulose (MC)-based 3D hPGCLC induction system were first compared with that of a conventional U96 system. Then, we examined the gene expression of germ cell marker genes and the key epigenetic modifications of the EpCAM-/INTEGRINα6-high cells from the 3D MC induction system and the U96 system via quantitative PCR and immunofluorescence. Finally, the reliability of the MC-based 3D hPGCLC induction system was evaluated via the generation of induced pluripotent stem cells (iPSCs) from the testicular cells of one patient with obstructive azoospermia (OA) and followed by the subsequent differentiation of iPSCs into the germ cell lineage. Results In the present study, we demonstrated that the 3D MC induction system combined with low-cell attachment plates facilitated the generation of hPGCLCs at a large scale. We found that the hPGCLCs generated via the MC system shared similar characteristics to that via the U96 system in terms of the gene expression profiles, germ cell-specific markers, epigenetic modification states and cellular states. In addition, hPGCLCs from iPSCs derived from one OA patient were generated with high efficiency via the present 3D MC induction system. Discussion The in vitro induction of hPGCLCs from human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs) has significant implications in exploring the underlying mechanisms of the origin and specification of hPGCs and the epigenetic programming of the human germ line as well as treating male infertility. Here, we developed a simple and efficient 3D induction system to generate hPGCLCs from hESCs/hiPSCs at a large scale, which facilitated the study of human germ cell development and stem cell-based reproductive medicine.
Wang et al. (2019) Efficient generation of human primordial germ cell-like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale. PeerJ 6:e6143 https://doi.org/10.7717/peerj.6143
Changes in bone density and bone quality caused by single fasting for 96 hours in rats
Background Young women occasionally engage in dietary restrictions accompanied by fasting for the purpose of losing weight, but such restrictions have various effects on body functions. The recent increase in the number of people suffering from osteoporosis has become a major social problem mainly in industrialized countries.Therefore, we think it is important to understand the effects of fasting on bone vulnerability, especially to bone quality. Methods Animals used male Wister rats weighing 130 g (6 weeks of age), and were divided into a control group (n = 5) and a fasting group (n = 6). The experimental period was 14 days, the control group had ad libitum food throughout the experimental period, the fasted group was fasted for 4 days, and than, had ad libitum food for 10 days. In this study, parameters related to bone fragility due to three-dimensional bone architecture were determined on Contrast enhanced micro-CT images of the lumbar spine and were used as a method for the evaluation of bone quality. In addition, a time-course observation of each individual was carried out during the fasting period and later upon resuming food intake. Cross-sectional images of all vertebrae were obtained from radiographic computed tomography and were analyzed by using Latheta software ver. 3.0 (Hitachi-Aloka Medical, Nagasaki, Japan). The region of interest that was misrecognized in each cross-sectional image was made consistent with the anatomical structure by carrying out corrections manually and by identifying the cortical bone areas and cancellous bone areas. Results Our findings showed that while single fasting for 96 h did not cause any major change in the macroscopic morphology of bone, it caused a marked decrease in bone density. In addition, the minimum cross-sectional moment, which indicated the “strength against bending” as well as the polar moment that indicated the “strength against torsion” were both lower than in non-fasted rats. Further, after resumption of feeding, bone mineral content in the fasting group recovered rapidly and starting at day 4 after resumption of feeding, there was no difference with the control group. On the other hand, the values of the minimum cross-sectional moment and polar moment did not recover, and the difference with the control group increased during the feeding period. Discussion On the basis of this study, the authors estimate that the fasting-induced decrease in bone minimum cross-sectional moment and polar moment may have been due to changes affecting some factors involved in bone quality, and thus could be useful as a parameter in future studies aimed at elucidating bone quality. At least, in the case where bone change accompanied with a change in macroscopic distribution of mineral components occurs, the values of minimum cross-sectional moment and polar moment are considered to be bone parameters that will provide valuable information to elucidate bone quality.
Hisatomi et al. (2019) Changes in bone density and bone quality caused by single fasting for 96 hours in rats. PeerJ 6:e6161 https://doi.org/10.7717/peerj.6161
Microbiota variations in Culex nigripalpus disease vector mosquito of West Nile virus and Saint Louis Encephalitis from different geographic origins
Although mosquito microbiota are known to influence reproduction, nutrition, disease transmission, and pesticide resistance, the relationship between host-associated microbial community composition and geographical location is poorly understood. To begin addressing this knowledge gap, we characterized microbiota associated with adult females of Culex nigripalpus mosquito vectors of Saint Louis Encephalitis and West Nile viruses sampled from three locations in Florida (Vero Beach, Palmetto Inland, and Palmetto Coast). High-throughput sequencing of PCR-amplified 16S rRNA genes demonstrated significant differences among microbial communities of mosquitoes sampled from the three locations. Mosquitoes from Vero Beach (east coast Florida) were dominated by uncultivated Asaia sp. (Alphaproteobacteria), whereas microbiota associated with mosquitoes collected from two mosquito populations at Palmetto (west coast Florida) sites were dominated by uncultured Spironema culicis (Spirochaetes), Salinisphaera hydrothermalis (Gammaproteobacteria), Spiroplasma (Mollicutes), uncultured Enterobacteriaceae, Candidatus Megaira (Alphaproteobacteria; Rickettsiae), and Zymobacter (Gammaproteobacteria). The variation in taxonomic profiles of Cx. nigripalpus gut microbial communities, especially with respect to dominating taxa, is a potentially critical factor in understanding disease transmission and mosquito susceptibility to insecticides among different mosquito populations.
Duguma et al. (2019) Microbiota variations in Culex nigripalpus disease vector mosquito of West Nile virus and Saint Louis Encephalitis from different geographic origins. PeerJ 6:e6168 https://doi.org/10.7717/peerj.6168
Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay
Blue mold is a postharvest rot of pomaceous fruits caused by Penicillium expansum and a number of other Penicillium species. The genome of the highly aggressive P. expansum strain R19 was re-sequenced and analyzed together with the genome of the less aggressive P. solitum strain RS1. Whole genome scale similarities and differences were examined. A phylogenetic analysis of P. expansum, P. solitum, and several closely related Penicillium species revealed that the two pathogens isolated from decayed apple with blue mold symptoms are not each other’s closest relatives. Among a total of 10,560 and 10,672 protein coding sequences respectively, a comparative genomics analysis revealed 41 genes in P. expansum R19 and 43 genes in P. solitum RS1 that are unique to these two species. These genes may be associated with pome fruit–fungal interactions, subsequent decay processes, and mycotoxin accumulation. An intact patulin gene cluster consisting of 15 biosynthetic genes was identified in the patulin producing P. expansum strain R19, while only a remnant, seven-gene cluster was identified in the patulin-deficient P. solitum strain. However, P. solitum contained a large number of additional secondary metabolite gene clusters, indicating that this species has the potential capacity to produce an array of known as well as not-yet-identified products of possible toxicological or biotechnological interest.
Wu et al. (2019) Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay. PeerJ 7:e6170 https://doi.org/10.7717/peerj.6170
Social network analysis for the assessment of pig, cattle and buffalo movement in Xayabouli, Lao PDR
The aim of this study is to understand the role that the movement patterns of pigs, cattle and buffalo play in the spread of foot-and-mouth disease (FMD). A cross-sectional survey consisting of a questionnaire was used in a hotspot area for FMD: Xayabouli Province, Lao People’s Democratic Republic. A total of 189 respondents were interviewed. We found that the key players in this network were people who were involved with more than one species of animal or occupation (multipurpose occupational node), which represents the highest number of activities of animals moved off the holding (shown with the highest out-degree centrality) and a high likelihood of being an intermediary between others (shown with the highest betweenness centrality). Moreover, the results show that the animals moved to and away from each node had few connections. Some nodes (such as traders) always received animals from the same group of cattle owners at different times. The subgroup connection within this network has many weak components, which means a connection in this network shows that some people can be reached by others, but most people were not. In this way, the number of connections present in the network was low when we defined the proportion of observed connections with all possible connections (density). These findings indicate that the network might not be busy; only one type of node is dominant which enables increased control of disease spread. We recommend that the relevant authorities implement control measures regarding the key players, which is the best way to effectively control the spread of infectious diseases.
Poolkhet et al. (2019) Social network analysis for the assessment of pig, cattle and buffalo movement in Xayabouli, Lao PDR. PeerJ 6:e6177 https://doi.org/10.7717/peerj.6177
A public database for the new MLST scheme for Treponema pallidum subsp. pallidum: surveillance and epidemiology of the causative agent of syphilis
Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with worldwide prevalence. Several different molecular typing schemes are currently available for this pathogen. To enable population biology studies of the syphilis agent and for epidemiological surveillance at the global scale, a harmonized typing tool needs to be introduced. Recently, we published a new multi-locus sequence typing (MLST) with the potential to significantly enhance the epidemiological data in several aspects (e.g., distinguishing genetically different clades of syphilis, subtyping inside these clades, and finally, distinguishing different subspecies of non-cultivable pathogenic treponemes). In this short report, we introduce the PubMLST database for treponemal DNA data storage and for assignments of allelic profiles and sequencing types. Moreover, we have summarized epidemiological data of all treponemal strains (n = 358) with available DNA sequences in typing loci and found several association between genetic groups and characteristics of patients. This study proposes the establishment of a single MLST of T. p. pallidum and encourages researchers and public health communities to use this PubMLST database as a universal tool for molecular typing studies of the syphilis pathogen.
Grillova et al. (2019) A public database for the new MLST scheme for Treponema pallidum subsp. pallidum: surveillance and epidemiology of the causative agent of syphilis. PeerJ 6:e6182 https://doi.org/10.7717/peerj.6182
MLPA is a practical and complementary alternative to CMA for diagnostic testing in patients with autism spectrum disorders and identifying new candidate CNVs associated with autism
Background Autism spectrum disorder (ASD) is a complex heterogeneous developmental disease with a significant genetic background that is frequently caused by rare copy number variants (CNVs). Microarray-based whole-genome approaches for CNV detection are widely accepted. However, the clinical significance of most CNV is poorly understood, so results obtained using such methods are sometimes ambiguous. We therefore evaluated a targeted approach based on multiplex ligation-dependent probe amplification (MLPA) using selected probemixes to detect clinically relevant variants for diagnostic testing of ASD patients. We compare the reliability and efficiency of this test to those of chromosomal microarray analysis (CMA) and other tests available to our laboratory. In addition, we identify new candidate genes for ASD identified in a cohort of ASD-diagnosed patients. Method We describe the use of MLPA, CMA, and karyotyping to detect CNV in 92 ASD patients and evaluate their clinical significance. Result Pathogenic and likely pathogenic mutations were identified by CMA in eight (8.07% of the studied cohort) and 12 (13.04%) ASD patients, respectively, and in eight (8.07%) and four (4.35%) patients, respectively, by MLPA. The detected mutations include the 22q13.3 deletion, which was attributed to ring chromosome 22 formation based on karyotyping. CMA revealed a total of 91 rare CNV in 55 patients: eight pathogenic, 15 designated variants of unknown significance (VOUS)—likely pathogenic, 10 VOUS—uncertain, and 58 VOUS—likely benign or benign. MLPA revealed 18 CNV in 18 individuals: eight pathogenic, four designated as VOUS—likely pathogenic, and six designated as VOUS—likely benign/benign. Rare CNVs were detected in 17 (58.62%) out of 29 females and 38 (60.32%) out of 63 males in the cohort. Two genes, DOCK8 and PARK2, were found to be overlapped by CNV designated pathogenic, VOUS—likely pathogenic, or VOUS—uncertain in multiple patients. Moreover, the studied ASD cohort exhibited significant (p < 0.05) enrichment of duplications encompassing DOCK8. Conclusion Multiplex ligation-dependent probe amplification and CMA yielded concordant results for 12 patients bearing CNV designated pathogenic or VOUS—likely pathogenic. Unambiguous diagnoses were achieved for eight patients (corresponding to 8.7% of the total studied population) by both MLPA and CMA, for one (1.09%) patient by karyotyping, and for one (1.09%) patient by FRAXA testing. MLPA and CMA thus achieved identical reliability with respect to clinically relevant findings. As such, MLPA could be useful as a fast and inexpensive test in patients with syndromic autism. The detection rate of potentially pathogenic variants (VOUS—likely pathogenic) achieved by CMA was higher than that for MLPA (13.04% vs. 4.35%). However, there was no corresponding difference in the rate of unambiguous diagnoses of ASD patients. In addition, the results obtained suggest that DOCK8 may play a role in the etiology of ASD.
Capkova et al. (2019) MLPA is a practical and complementary alternative to CMA for diagnostic testing in patients with autism spectrum disorders and identifying new candidate CNVs associated with autism. PeerJ 6:e6183 https://doi.org/10.7717/peerj.6183
First record of Phyllorhiza sp. (Cnidaria: Scyphozoa) in a Chinese coastal aquaculture pond
Background It has been suggested that aquaculture ponds on the Chinese coast could act as breeding grounds for scyphozoans. Here, we present the first record of the scyphomedusa Phyllorhiza sp. in an aquaculture pond on the coast of the southern Yellow Sea, based on a combination of morphological characteristics and mitochondrial 16S DNA sequence data. Methods A field survey was performed on June 29, 2017 in a pond used for culturing the shrimp Penaeus japonicus, located in the southern Yellow Sea, China. Jellyfish specimens were collected for morphological and genetic analysis. The morphological characters of the jellyfish specimens were compared to taxonomic literature. Additionally, phylogenetic analysis of the mitochondrial 16S fragments of these specimens were also conducted. Results These specimens had the following morphological characters: hemispherical umbrella without scapulets; J-shaped oral arms; a single larger terminal club on each arm; bluish colored with a slightly expanded white tip; and mouthlets present only in the lower half to one-third of each arm. These morphological features of the medusae indicated that the specimens found in the shrimp culture ponds belong to the genus Phyllorhiza Agassiz, 1862, but did not match with the description of any of the known species of the genus Phyllorhiza. Phylogenetic analyses of the mtDNA 16S regions revealed that these specimens, together with Phyllorhiza sp. from Malaysian coastal waters, belong to a sister group of Phyllorhiza punctata. Juveniles and ephyrae of Phyllorhiza sp. were observed in the aquaculture pond. The mean density of Phyllorhiza sp. medusa in the surface water within the pond was estimated to be 0.05 individuals/m2. Discussion Based on our observations of the gross morphology and molecular data, we state that the specimens collected in the aquaculture pond can be identified as Phyllorhiza sp. This is the first record of Phyllorhiza sp. in Chinese seas. Large scale dispersal through ballast water or the expansion of jellyfish aquarium exhibitions are possible pathways of invasion, but this needs to be confirmed in further studies.
Dong et al. (2019) First record of Phyllorhiza sp. (Cnidaria: Scyphozoa) in a Chinese coastal aquaculture pond. PeerJ 7:e6191 https://doi.org/10.7717/peerj.6191
Climatic niche evolution in the viviparous Sceloporus torquatus group (Squamata: Phrynosomatidae)
The cold-climate hypothesis maintains that viviparity arose as a means to prevent increased egg mortality in nests owing to low temperatures, and this hypothesis represents the primary and most strongly supported explanation for the evolution of viviparity in reptiles. In this regard, certain authors have stated that viviparous species will exhibit speciation via climatic niche conservatism, with similar climatic niches being observed in allopatric sister species. However, this prediction remains to be tested with bioclimatic variables relevant to each viviparous group. In the present study, we examined climatic niche evolution in a group of North American viviparous lizards to determine whether their diversification is linked to phylogenetic niche conservatism (PNC). We evaluated the phylogenetic signal and trait evolution of individual bioclimatic variables and principal component (PC) scores of a PC analysis, along with reconstructions of ancestral climate tolerances. The results suggest that diversification of the Sceloporus torquatus group species is associated with both niche differentiation and PNC. Furthermore, we did not observe PNC across nearly all bioclimatic variables and in PC2 and PC3. However, in Precipitation Seasonality (Bio15), in Precipitation of Coldest Quarter (Bio19) and in PC1 (weakly associated with variability of temperature), we did observe PNC. Additionally, variation of the scores along the phylogeny and Pagel’s delta (δ) >1 of PC3 suggests a fast, recent evolution to dry conditions in the clade that sustains S. serrifer.
Martínez-Méndez et al. (2019) Climatic niche evolution in the viviparous Sceloporus torquatus group (Squamata: Phrynosomatidae). PeerJ 6:e6192 https://doi.org/10.7717/peerj.6192
Carabid community structure in northern China grassland ecosystems: Effects of local habitat on species richness, species composition and functional diversity
Background Most carabid beetles are particularly sensitive to local habitat characteristics. Although in China grasslands account for more than 40% of the national land, their biodiversity is still poorly known. The aim of this paper is to identify the main environmental characteristics influencing carabid diversity in different types of grassland in northern China. Methods We investigated the influence of vegetation (plant biomass, cover, density, height and species richness), soil (bulk density, above ground litter, moisture and temperature) and climate (humidity, precipitation and temperature) on carabid community structure (species richness, species composition and functional diversity—measured as body size, movement and total diversity) in three types of grasslands: desert, typical and meadow steppes. We used Canonical correspondence analysis to investigate the role of habitat characteristics on species composition and eigenvector spatial filtering to investigate the responses of species richness and functional diversities. Results We found that carabid community structure was strongly influenced by local habitat characteristics and particularly by climatic factors. Carabids in the desert steppe showed the lowest richness and functional diversities. Climate predictors (temperature, precipitation and humidity) had positive effects on carabid species richness at both regional and ecosystem levels, with difference among ecosystems. Plant diversity had a positive influence on carabid richness at the regional level. Soil compaction and temperature were negatively related to species richness at regional level. Climatic factors positively influenced functional diversities, whereas soil temperature had negative effects. Soil moisture and temperature were the most important drivers of species composition at regional level, whereas the relative importance of the various environmental parameters varied among ecosystems. Discussion Carabid responses to environmental characteristics varied among grassland types, which warns against generalizations and indicates that management programs should be considered at grassland scale. Carabid community structure is strongly influenced by climatic factors, and can therefore be particularly sensitive to ongoing climate change.
Tsafack et al. (2019) Carabid community structure in northern China grassland ecosystems: Effects of local habitat on species richness, species composition and functional diversity. PeerJ 6:e6197 https://doi.org/10.7717/peerj.6197