Genome-wide Cas9 binding specificity in Saccharomyces cerevisiae
The CRISPR system has become heavily utilized in biomedical research as a tool for genomic editing as well as for site-specific chromosomal localization of specific proteins. For example, we developed a CRISPR-based methodology for enriching a specific genomic locus of interest for proteomic analysis in Saccharomyces cerevisiae, which utilized a guide RNA-targeted, catalytically dead Cas9 (dCas9) as an affinity reagent. To more comprehensively evaluate the genomic specificity of using dCas9 as a site-specific tool for chromosomal studies, we performed dCas9-mediated locus enrichment followed by next-generation sequencing on a genome-wide scale. As a test locus, we used the ARS305 origin of replication on chromosome III in S. cerevisiae. We found that enrichment of this site is highly specific, with virtually no off-target enrichment of unique genomic sequences. The high specificity of genomic localization and enrichment suggests that dCas9-mediated technologies have promising potential for site-specific chromosomal studies in organisms with relatively small genomes such as yeasts.
Waldrip et al. (2020) Genome-wide Cas9 binding specificity in Saccharomyces cerevisiae. PeerJ 8:e9442 https://doi.org/10.7717/peerj.9442
Evaluation of knowledge, attitudes, and clinical education of dental students about COVID-19 pandemic
Background The novel coronavirus disease (COVID-19) is a new viral respiratory illness, first identified in Wuhan province, China. Dental professionals and dental students are at an increased risk for these viruses from dental patients, as dental practice involves face-to-face communication with the patients and frequent exposure to saliva, blood, and other body fluids. Dental education can play an important role in the training of dental students, adequate knowledge and adopting attitudes regarding infection control measures. The aim of this study was to evaluate knowledge, attitudes, and clinical education of dental students about COVID-19 pandemic. Methods A total of 355 pre-clinical and clinical dental students (242 and 113, respectively, comprising 190 females and 165 males) at Fırat University Dentistry Faculty, in Elazığ, Turkey answered an online questionnaire about the biosafety procedures for and their attitudes to and knowledge of COVID-19. The study was conducted in March 2020, Turkey. The data gained were analyzed using descriptive statistical methods and chi-square test. Results Both the clinical and preclinical students were found to be afraid of infecting themselves and their environment with COVID-19, and the difference between them was statistically significant. Three quarters (74.9%) of the participants responded yes to the question of whether they thought that experiences related to COVID-19 affected them psychologically, with the differences between gender and clinical status were statistically significant. Responses to the question of which clinical rotation worried them more were 29.9% endodontics, 25.1% oral and maxillofacial surgery, 16.3% prosthesis, 15.2% periodontology, 6.8% restorative dentistry, 3.9% oral diagnosis and radiology, 1.7% pedodontics, and 1.1% orthodontics, with a significant difference between the preclinical and clinical students. Regarding the measures applied by the clinical students in their clinical rotation, the responses were 100% gloves and 100% mask (with 11.5% FFP3/N95 mask), 73.6% face protective shield and 37.1% safety glasses, and 49% bonnet and 16.8% disposable box, with 90.2% frequent hand washing, and 86.7% frequent hand antiseptic usage. Conclusions While students gave good responses regarding the standard measures they take to protect against transmission of COVID-19, their knowledge and attitudes about the extra measures they can take should be improved. For students to be least affected by fears associated with the disease, dental faculties should be ready to provide psychological services to those in need.
Ataş et al. (2020) Evaluation of knowledge, attitudes, and clinical education of dental students about COVID-19 pandemic. PeerJ 8:e9575 https://doi.org/10.7717/peerj.9575
Altered gut microbiota associated with symptom severity in schizophrenia
Background The gut microbiome and microbiome-gut-brain (MGB) axis have been receiving increasing attention for their role in the regulation of mental behavior and possible biological basis of psychiatric disorders. With the advance of next-generation sequencing technology, characterization of the gut microbiota in schizophrenia (SZ) patients can provide rich clues for the diagnosis and prevention of SZ. Methods In this study, we compared the differences in the fecal microbiota between 82 SZ patients and 80 demographically matched normal controls (NCs) by 16S rRNA sequencing and analyzed the correlations between altered gut microbiota and symptom severity. Results The alpha diversity showed no significant differences between the NC and SZ groups, but the beta diversity revealed significant community-level separation in microbiome composition between the two groups (pseudo-F =3.337, p < 0.001, uncorrected). At the phylum level, relatively more Actinobacteria and less Firmicutes (p < 0.05, FDR corrected) were found in the SZ group. At the genus level, the relative abundances of Collinsella, Lactobacillus, Succinivibrio, Mogibacterium, Corynebacterium, undefined Ruminococcus and undefined Eubacterium were significantly increased, whereas the abundances of Adlercreutzia, Anaerostipes, Ruminococcus and Faecalibacterium were decreased in the SZ group compared to the NC group (p < 0.05, FDR corrected). We performed PICRUSt analysis and found that several metabolic pathways differed significantly between the two groups, including the Polyketide sugar unit biosynthesis, Valine, Leucine and Isoleucine biosynthesis, Pantothenate and CoA biosynthesis, C5-Branched dibasic acid metabolism, Phenylpropanoid biosynthesis, Ascorbate and aldarate metabolism, Nucleotide metabolism and Propanoate metabolism pathways (p < 0.05, FDR corrected). Among the SZ group, the abundance of Succinivibrio was positively correlated with the total Positive and Negative Syndrome Scale (PANSS) scores (r = 0.24, p < 0.05, uncorrected) as well as the general PANSS scores (r = 0.22, p < 0.05, uncorrected); Corynebacterium was negatively related to the negative scores of PANSS (r = 0.22, p < 0.05, uncorrected). Conclusions Our findings provided evidence of altered gut microbial composition in SZ group. In addition, we found that Succinvibrio and Corynebacterium were associated with the severity of symptoms for the first time, which may provide some new biomarkers for the diagnosis of SZ.
Li et al. (2020) Altered gut microbiota associated with symptom severity in schizophrenia. PeerJ 8:e9574 https://doi.org/10.7717/peerj.9574
Identification of viruses infecting six plum cultivars in Korea by RNA-sequencing
Background Plums are a kind of stone fruit, a category that includes peaches, cherries, apricots, and almonds. In Korea, Japanese plum trees are usually cultivated as they best suit the climate. To date, there have been few studies in Korea on viruses infecting plum trees compared to those infecting peach trees. Methods To identify viruses and viroids infecting plum trees, we collected leaf samples from six different plum cultivars and subjected them to RNA-sequencing (RNA-seq). Six different plum transcriptomes were de novo assembled using the Trinity assembler followed by BLAST searching against a viral reference database. Results We identified hop stunt viroid (HSVd) and six viruses, including apple chlorotic leaf spot virus (ACLSV), little cherry virus-1 (LChV-1), peach virus D (PeVD), peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and prunus necrotic ringspot virus (PNRSV), from six plum cultivars by RNA-seq. RT-PCR confirmed the infection of HSVd and three viruses—ACLSV, PBNSPaV, and PNRSV—in plum trees. However, RT-PCR demonstrated that plum trees in this study were not infected by LChV-1, PeVD, or PLPaV. It is likely that the three viruses LChV-1, PeVD, and PLPaV as identified by RNA-seq were contaminants from other peach libraries caused by index misassignment, which suggests that careful confirmation by other methods should be carried out in next-generation sequencing (NGS)-based virus identification. Taken together, we identified a viroid and three viruses infecting plum trees in Korea.
Jo et al. (2020) Identification of viruses infecting six plum cultivars in Korea by RNA-sequencing. PeerJ 8:e9588 https://doi.org/10.7717/peerj.9588
Assessment of productivity, nutrient uptake and economic benefits of rice under different nitrogen management strategies
Background Integrating a chemical nitrogen (N) fertilizer with an organic fertilizer and using slow-release mechanism are important N management strategies to increase the N utilization efficiency (NUE) and grain yield of rice. However, the performances of both N management strategies on the productivity, the nutrient absorption and utilization efficiency, and the economic benefits of rice have not yet been comprehensively evaluated. Methods A 2-year field experiment was conducted with seven N management strategies without fertilizer (control), 100% conventional N fertilizer (conventional compound fertilizer and urea) (N100), 75% conventional N fertilizer with 25% organic–inorganic compound fertilizer (N75+OICF25), 50% conventional N fertilizer with 50% organic–inorganic compound fertilizer (N50+OICF50), 100% organic–inorganic compound fertilizer (OICF100), slow-release compound fertilizer with urea (SRCF+U), compound fertilizer with sulfur-coated urea (CF+SCU). The responses of the productivity, the nutrient absorption and utilization efficiency, and the economic benefits of rice to the different N management strategies were evaluated. Results CF+SCU performed comparably or better than N100, judging by the grain yield (GY), the N, phosphate (P) and potassium (K) agronomic efficiency (NAE, PAE and KAE), and the apparent N, P and K recovery efficiency (ANRE, APRE and AKRE). SRCF+U significantly increased the GY by an average of 7.7%, the NAE and the ANRE by 23.8 and 26.7%, the PAE and the APRE by 90.6 and 109.3%, and the KAE and the AKRE by 74.2 and 57.7%. The higher GY and nutrient utilization efficiency when using SRCF+U were attributed to the higher total biomass and total nutrient absorption. N75+OICF25 and N50+OICF50 produced a comparable grain yield than N100, whereas a significant yield reduction was observed when using OICF100. Compared with N100, N75+OICF25 resulted in a comparable or higher fertilizer use efficiency (0.3 and 4.7% for NAE and ANRE, 0.3 and 3.2% for PAE and APRE, 0.3 and −2.8% for KAE and AKRE). However, the fertilizer use efficiency when using N50+OICF50 and OICF100 were lower than with N100. The highest net return (NR) (5,845.03 yuan ha−1) and benefit to cost (B:C) ratio (0.34) were obtained when using SRCF+U. The NR and the B:C ratio when using N75+OICF25 were slightly higher than when using N100. However, N50+OICF50 and OICF100 significantly decreased the NR and the B:C ratio compared with N100 by 14.5 and 12.1% and by 35.1 and 29.0%, respectively. Conclusions SRCF+U and CF+SCU enhanced the crop productivity, the nutrient uptake and utilization efficiency, and the economic benefits compared with N100. The comprehensive performance of SRCF+U was better than that of CF+SCU. N75+OICF25 produced almost similar productivity, nutrient uptake and use efficiency compared with N100. It demonstrated that N75+OICF25 stabilized the grain yield production of rice and reduced the input of chemical N fertilizer.
Yang et al. (2020) Assessment of productivity, nutrient uptake and economic benefits of rice under different nitrogen management strategies. PeerJ 8:e9596 https://doi.org/10.7717/peerj.9596
The diet of otters (Lutra lutra) on the Agri river system, one of the most important presence sites in Italy: a molecular approach
Background The Eurasian otter (Lutra lutra) underwent a strong decline in large areas of the Central-Western part of its distribution range, during the second half of the twentieth century. In Italy, only residual fragmented nuclei survive in the Central-Southern part of the peninsula. Nowadays, the otter is one of the most endangered mammals in Italy, and increasing the knowledge about the ecology of this species is a key step in defining fitting management strategies. Here we provide information about the diet of otter on the Agri river system, one of the most important presence sites in Italy, to understand both the species’ food requirements and the impact on fish communities. Methods DNA metabarcoding and High Throughput Sequencing were used on DNA extracted from spraints. We amplified DNA with a primer set for vertebrates, focusing efforts on the bulk of the otter’s diet (fishes and amphibians). Results Our findings showed that the diet of the otter was dominated by cyprinids (97.77%, and 99.14% of fishes), while amphibians represented 0.85% of the sequences analyzed. Results are in general accordance with previous studies based on morphological characterization; however, molecular analyses allow the resolving of some morphological uncertainties. Although the study area offers a very wide range of available prey, the diet of the otters shows marked selectivity. We highlighted a variation in prey consumed, in accordance with the typology of water system (i.e., river, lake, tributary). Some of the preys found in the diet were alien species introduced by man for sport fishing. Our findings could help define strategies useful for the conservation of the otter population in Southern Italy, suggesting management actions directed at avoiding fish community alterations through illegal stockings without severe controls on their taxonomic status. These introductions could result in a general reduction in the diversity of the otter’s preys, affecting its predatory behavior.
Buglione et al. (2020) The diet of otters (Lutra lutra) on the Agri river system, one of the most important presence sites in Italy: a molecular approach. PeerJ 8:e9606 https://doi.org/10.7717/peerj.9606
Metagenomic next-generation sequencing for the clinical diagnosis and prognosis of acute respiratory distress syndrome caused by severe pneumonia: a retrospective study
Background Metagenome next-generation sequencing (mNGS) is a valuable diagnostic tool that can be used for the identification of early pathogens of acute respiratory distress syndrome (ARDS) in severe pneumonia. Little is known about the use of this technology in clinical application and the evaluation of the prognostic value of ARDS. Methods We performed a retrospective cohort study of patients with ARDS caused by severe pneumonia. Samples were collected from patients in the intensive care unit (ICU) of Jiangmen Central Hospital from January 2018 to August 2019. The no-next generation sequencing (NGS) group was composed of patients given conventional microbiological tests to examine sputum, blood, or bronchoalveolar lavage fluid. The NGS group was composed of patients tested using mNGS and conventional microbiological tests. We evaluated the etiological diagnostic effect and clinical prognostic value of mNGS in patients with ARDS caused by severe pneumonia. Results The overall positive rate (91.1%) detected by the mNGS method was significantly higher than that of the culture method (62.2%, P = 0.001), and antibody plus polymerase chain reaction (28.9%, P < 0.001). Following adjustment of the treatment plan based on microbial testing results, the Acute Physiology and Chronic Health Evaluation-II (APACHE II) score of the NGS group was lower than that of the no-NGS group 7 days after treatment (P < 0.05). The 28-day mortality rate of the NGS group was significantly lower than that of the no-NGS group (P < 0.05). Longer ICU stay, higher APACHE II score and sequential organ failure assessment score were risk factors for the death of ARDS, and adjusting the medication regimen based on mNGS results was a protective factor. The detection of mNGS can significantly shorten the ICU stay of immunosuppressed patients (P < 0.01), shorten the ventilation time (P < 0.01), and reduce the ICU hospitalization cost (P < 0.05). Conclusions Metagenome next-generation sequencing is a valuable tool to determine the etiological value of ARDS caused by severe pneumonia to improve diagnostic accuracy and prognosis for this disease. For immunosuppressed patients, mNGS technology can be used in the early stage to provide more diagnostic evidence and guide medications.
Zhang et al. (2020) Metagenomic next-generation sequencing for the clinical diagnosis and prognosis of acute respiratory distress syndrome caused by severe pneumonia: a retrospective study. PeerJ 8:e9623 https://doi.org/10.7717/peerj.9623
Spermatogenesis is normal in Tex33 knockout mice
Testis expressed gene 33 (Tex33) is a recently reported testis-specific gene and it is evolutionarily conserved in vertebrates. The Tex33 expression is found in cytoplasm of round spermatids in Mus musculus. However, the in vivo function of Tex33 remains unknown. In this study, we made a 62bp in frame deletion on Exon2 of Tex33 gene by CRISPR/Cas9 in C57B/L6 mouse, which cause frame shift mutation of Tex33 gene. Tex33-/-adult male were fertile, and there is no significant change on litter size compared with male wildtype (Tex33+/+) adult. Besides, no overt differences were found in testis/body weight ratios, testicular/epididymal tissue morphology, sperm counts, sperm morphology and spermatozoa motility in adult Tex33-/-male mice (N = 3), in comparison with Tex33+/+ adult (N = 3). TUNEL assay also indicates the germ cells apoptosis ratio was not significantly changed in adult Tex33-/- adult male mouse testis (N = 3), compared with adult Tex33+/+ male (N = 3). Importantly, the first wave of elongating spermatids formation happens in 5w old mice. We find that the first wave of spermiogenesis is not disrupted in both 5-week-old Tex33+/+ and Tex33-/-male mouse testes and three hallmarks of spermatogenesis, PLZF,γ-H2AX and TNP1, are all detectable in seminiferous tubule. All results indicate that Tex33 is a redundant gene to spermatogenesis. This study can help other researchers avoid repetitive works on redundant genes.
Zhu et al. (2020) Spermatogenesis is normal in Tex33 knockout mice. PeerJ 8:e9629 https://doi.org/10.7717/peerj.9629
Five genes influenced by obesity may contribute to the development of thyroid cancer through the regulation of insulin levels
Previous studies indicate that obesity is an important contributor to the proceeding of thyroid cancer (TC) with limited knowledge of the underlying mechanism. Here, we hypothesize that molecules affected by obesity may play roles in the development of TC. To test the hypothesis above, we first conducted a large-scale literature-based data mining to identify genes influenced by obesity and genes related to TC. Then, a mega-analysis was conducted to study the expression changes of the obesity-specific genes in the case of TC, using 16 independent TC array-expression datasets (783 TC cases and 439 healthy controls). After that, pathway analysis was performed to explore the functional profile of the selected target genes and their potential connections with TC. We identified 1,036 genes associated with TC and 534 regulated by obesity, demonstrating a significant overlap (N = 176, p-value = 4.07e−112). Five out of the 358 obesity-specific genes, FABP4, CFD, GHR, TNFRSF11B, and LTF, presented significantly decreased expression in TC patients (LFC<−1.44; and p-value < 1e−7). Multiple literature-based pathways were identified where obesity could promote the pathologic development of TC through the regulation of these five genes and INS levels. The five obesity genes uncovered could be novel genes that play roles in the etiology of TC through the modulation of INS levels.
Chen et al. (2020) Five genes influenced by obesity may contribute to the development of thyroid cancer through the regulation of insulin levels. PeerJ 8:e9302 https://doi.org/10.7717/peerj.9302
Assessment of potato peel and agro-forestry biochars supplementation on in vitro ruminal fermentation
Background The awareness of environmental and socio-economic impacts caused by greenhouse gas emissions from the livestock sector leverages the adoption of strategies to counteract it. Feed supplements can play an important role in the reduction of the main greenhouse gas produced by ruminants—methane (CH4). In this context, this study aims to assess the effect of two biochar sources and inclusion levels on rumen fermentation parameters in vitro. Methods Two sources of biochar (agro-forestry residues, AFB, and potato peel, PPB) were added at two levels (5 and 10%, dry matter (DM) basis) to two basal substrates (haylage and corn silage) and incubated 24-h with rumen inocula to assess the effects on CH4 production and main rumen fermentation parameters in vitro. Results AFB and PPB were obtained at different carbonization conditions resulting in different apparent surface areas, ash content, pH at the point of zero charge (pHpzc), and elemental analysis. Relative to control (0% biochar), biochar supplementation kept unaffected total gas production and yield (mL and mL/g DM, p = 0.140 and p = 0.240, respectively) and fermentation pH (p = 0.666), increased CH4production and yield (mL and mL/g DM, respectively, p = 0.001) and ammonia-N (NH3-N, p = 0.040), and decreased total volatile fatty acids (VFA) production (p < 0.001) and H2 generated and consumed (p ≤ 0.001). Biochar sources and inclusion levels had no negative effect on most of the fermentation parameters and efficiency. Acetic:propionic acid ratio (p = 0.048) and H2 consumed (p = 0.019) were lower with AFB inclusion when compared to PPB. Biochar inclusion at 10% reduced H2 consumed (p < 0.001) and tended to reduce total gas production (p = 0.055). Total VFA production (p = 0.019), acetic acid proportion (p = 0.011) and H2 generated (p = 0.048) were the lowest with AFB supplemented at 10%, no differences being observed among the other treatments. The basal substrate affected most fermentation parameters independently of biochar source and level used. Discussion Biochar supplementation increased NH3-N content, iso-butyric, iso-valeric and valeric acid proportions, and decreased VFA production suggesting a reduced energy supply for microbial growth, higher proteolysis and deamination of substrate N, and a decrease of NH3-N incorporation into microbial protein. No interaction was found between substrate and biochar source or level on any of the parameters measured. Although AFB and PPB had different textural and compositional characteristics, their effects on the rumen fermentation parameters were similar, the only observed effects being due to AFB included at 10%. Biochar supplementation promoted CH4 production regardless of the source and inclusion level, suggesting that there may be other effects beyond biomass and temperature of production of biochar, highlighting the need to consider other characteristics to better identify the mechanism by which biochar may influence CH4 production.
Rodrigues et al. (2020) Assessment of potato peel and agro-forestry biochars supplementation on in vitro ruminal fermentation. PeerJ 8:e9488 https://doi.org/10.7717/peerj.9488