Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with ...varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5' end, allowing for a range of different 3' primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of
and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.
We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.
Bariatric surgery is highly effective for the treatment of obesity in individuals without (OB11OB: individuals with obesity.) and in those with type 2 diabetes (T2D22T2D: individuals with obesity and ...type 2 diabetes.). However, whether bariatric surgery triggers similar or distinct molecular changes in OB and T2D remains unknown. Given that individuals with type 2 diabetes often exhibit more severe metabolic deterioration, we hypothesized that bariatric surgery induces distinct molecular adaptations in skeletal muscle, the major site of glucose uptake, of OB and T2D after surgery-induced weight loss.
All participants (OB, n = 13; T2D, n = 13) underwent detailed anthropometry before and one year after the surgery. Skeletal muscle biopsies were isolated at both time points and subjected to transcriptome and methylome analyses using a comprehensive bioinformatic pipeline.
Before surgery, T2D had higher fasting glucose and insulin levels but lower whole-body insulin sensitivity, only glycemia remained higher in T2D than in OB after surgery. Surgery-mediated weight loss affected different subsets of genes with 2,013 differentially expressed in OB and 959 in T2D. In OB differentially expressed genes were involved in insulin, PPAR signaling and oxidative phosphorylation pathways, whereas ribosome and splicesome in T2D. LASSO regression analysis revealed distinct candidate genes correlated with improvement of phenotypic traits in OB and T2D. Compared to OB, DNA methylation was less affected in T2D in response to bariatric surgery. This may be due to increased global hydroxymethylation accompanied by decreased expression of one of the type 2 diabetes risk gene, TET2, encoding a demethylation enzyme in T2D.
OB and T2D exhibit differential skeletal muscle transcriptome responses to bariatric surgery, presumably resulting from perturbed epigenetic flexibility.
The mammalian pituitary gland is a complex organ consisting of hormone‐producing cells, anterior lobe folliculostellate cells (FSCs), posterior lobe pituicytes, vascular pericytes and endothelial ...cells, and Sox2‐expressing stem cells. We present single‐cell RNA sequencing and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on the transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe‐specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type‐specific. We also found that the SOX2 gene and protein were expressed in ~15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone‐producing cells, arguing against its stem cell specificity. FSCs comprised two Sox2‐expressing subclusters; FS1 contained more cells but lower genetic diversity, while FS2 contained proliferative cells, shared genes with hormone‐producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FS1 cells were randomly distributed in the anterior and intermediate lobes, while FS2 cells were localized exclusively in the marginal zone between the anterior and intermediate lobes. These data indicate the identity of the FSCs as anterior pituitary‐specific astroglia, with FS1 cells representing differentiated cells equipped for classical FSC roles and FS2 cells exhibiting additional stem cell‐like features.
Main Points
Pituitary folliculostellate cells (FSCs) were studied with scRNAseq and immunohistofluorescence.
Expression profiles of FSCs and pituicytes confirmed their astroglial identity.
FSC subtypes included pituitary stem‐like cells and differentiated FSCs.
Advances in high-throughput sequencing (HTS) have fostered rapid developments in the field of microbiome research, and massive microbiome datasets are now being generated. However, the diversity of ...software tools and the complexity of analysis pipelines make it difficult to access this field. Here, we systematically summarize the advantages and limitations of microbiome methods. Then, we recommend specific pipelines for amplicon and metagenomic analyses, and describe commonly-used software and databases, to help researchers select the appropriate tools. Furthermore, we introduce statistical and visualization methods suitable for microbiome analysis, including alpha- and betadiversity, taxonomic composition, difference comparisons, correlation, networks, machine learning, evolution, source tracing, and common visualization styles to help researchers make informed choices. Finally, a stepby-step reproducible analysis guide is introduced. We hope this review will allow researchers to carry out data analysis more effectively and to quickly select the appropriate tools in order to efficiently mine the biological significance behind the data.
High salinity, poor soil structure and nutrient deficiencies are the main limitations in saline-alkali soils. We investigated the effects on the microstructure, and the aerobic and anaerobic ...bacterial communities in the macroaggregates following addition of vermicompost and humic acid fertilizer to saline-alkali soil. These effects could enhance salt leaching and inhibit nitrogen losses in the topsoil. Vermicompost and humic acid fertilizer improved soil aggregate stability and the microstructure of macroaggregates (porosity, path tortuosity and effective porosity), which in turn increased soil permeability and salt leaching in saline-alkali topsoil. The improvement in aggregate microstructure affected the soil bacterial community of the macroaggregates by increasing the relative abundances of aerobic heterotrophs, such as Chloroflexi and Bacteroidetes. Additionally, vermicompost and humic acid fertilizer influenced nitrogen cycling by the macroaggregate microbial community, and stimulated nitrification by increasing archaeal and bacterial amoA gene copy numbers in the topsoil, but inhibited nitrite-reducing bacteria by decreasing nirS and nirK gene copy numbers. Furthermore, these amendments increased microbial N2O reduction by increasing nosZ gene copy numbers, suggesting that vermicompost and humic acid fertilizer have the potential to reduce N2O emissions in saline-alkali soil. In summary, vermicompost and humic acid fertilizer can ameliorate saline-alkali soils by replenishing organic matter, improving aggregate microstructure, and regulating the microbial community, which could increase salt leaching and inhibit nitrogen losses.
•Vermicompost (V) and humic acid fertilizer (H) improved aggregate microstructure in saline soil.•Improvement in aggregate microstructure affected soil bacterial community in macroaggregates.•Improvement in aggregate microstructure increased salt leaching and inhibited nitrogen loss.•V and H ameliorated saline soil by increasing SOM and improving aggregate microstructure.
Drought stress is the primary factor limiting the growth and fruit quality of grapevines worldwide. However, the biological function of the NAC No apical meristem (NAM), Arabidopsis transcription ...activation factor (ATAF), Cup-shaped cotyledon (CUC) transcription factor (TF) in grapevine is not clear. In this study, we reported that VvNAC17, a novel NAC transcription factor, was expressed in various tissues following drought, high temperature (45 °C), freezing (4 °C), salicylic acid (SA), and abscisic acid (ABA) treatments in grapevine. The VvNAC17 protein was localized in the nucleus of Arabidopsis thaliana protoplasts and demonstrated transcriptional activation activities at its C-terminus in yeast. The VvNAC17 gene was overexpressed in Arabidopsis thaliana. Under mannitol and salt stress, the germination rates of the VvNAC17-overexpression lines were higher than those of the wild-type plants, as were the root lengths. The VvNAC17-overexpression lines showed greater tolerance to freezing stress along with a higher survival rate. Following ABA treatment, the seed germination rate and the root length of the VvNAC17-overexpression lines were inhibited, and the stomatal opening and stomatal density were reduced. When subjected to drought and dehydration stress, the VvNAC17-overexpression lines showed improved survival and reduced water loss rates in comparison to the wild-type plants. Under drought conditions, the VvNAC17-overexpression lines had lower malondialdehyde and H2O2 contents, but higher peroxidase, superoxide dismutase, and catalase activities as well as higher proline content. Moreover, the expression of marker genes, including ABI5, AREB1, COR15A, COR47, P5CS, RD22, and RD29A, was up-regulated in the VvNAC17-overexpression lines when subjected to ABA and drought treatments. The results suggest that in transgenic Arabidopsis over-expression of VvNAC17 enhances resistance to drought while up-regulating the expression of ABA- and stress-related genes.
•VvNAC17 enhanced drought tolerance in Arabidopsis.•Overexpression of VvNAC17 in Arabidopsis plants showed increased abscisic acid sensitivity.•VvNAC17 was promoted in drought- and abscisic acid-induced stomatal closure.•Overexpression of VvNAC17 promoted the efficiency of enzymatic antioxidant defense systems in Arabidopsis.•Overexpression of VvNAC17 up-regulated ABA-signaling and stress-responsive marker genes in Arabidopsis.
An analysis was conducted on marker genes indicating the induction of clear cell renal cell carcinoma (ccRCC) and their role in the pathogenesis of this oncological disease. The diagnostic and ...therapeutic approaches for ccRCC are evolving with the identification of marker genes. The use of molecular biology methods has enabled the detection of somatic mutations in RCC marker genes like VHL, BAP1, OGG1, FLCN, MDM2, TP53, and RNF139. Targeted drugs like sorafenib, sunitinib, bevacizumab, pazopanib, axitinib, everolimus, and temsirolimus are being utilized in the treatment of RCC metastases. The diagnostic value of specific alleles in marker genes like rs1642742 and rs779805 of the VHL gene, rs1052133 of the OGG1 gene, rs2279744 of the MDM2 gene, and rs1597368777 of the TP53 gene has been confirmed in detecting ccRCC. Mutations in these marker genes play a significant role in the pathogenesis of ccRCC and are associated with the development and progression of the disease. The identification of these mutations can aid in the diagnosis, prognosis, and treatment of ccRCC. Marker genes are essential in understanding the molecular mechanisms underlying ccRCC. Mutations in genes like VHL, BAP1, OGG1, FLCN, MDM2, TP53, and RNF139 are key drivers of ccRCC pathogenesis. The diagnostic and therapeutic implications of these mutations are significant in the management of ccRCC. Further research into the role of marker genes in ccRCC is essential for developing targeted therapies and improving patient outcomes.
Natural killer (NK) cells, the effectors of the innate immune system, have a remarkable influence on cancer prognosis and immunotherapy. In this study, a total of 1,816 samples from nine independent ...cohorts in public datasets were enrolled. We first conducted a comprehensive analysis of single-cell RNA-sequencing data of lung adenocarcinoma (LUAD) from the Gene Expression Omnibus (GEO) database and determined 189 NK cell marker genes. Subsequently, we developed a seven-gene prognostic signature based on NK cell marker genes in the TCGA LUAD cohort, which stratified patients into high-risk and low-risk groups. The predictive power of the signature was well verified in different clinical subgroups and GEO cohorts. With a multivariate analysis, the signature was identified as an independent prognostic factor. Low-risk patients had higher immune cell infiltration states, especially CD8
+
T cells and follicular helper T cells. There existed a negative association between inflammatory activities and risk score, and the richness and diversity of the T-cell receptor (TCR) repertoire was higher in the low-risk groups. Importantly, analysis of an independent immunotherapy cohort (IMvigor210) revealed that low-risk patients had better immunotherapy responses and prognosis than high-risk patients. Collectively, our study developed a novel signature based on NK cell marker genes, which had a potent capability to predict the prognosis and immunotherapy response of LUAD patients.
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Advances in nucleic acid sequencing technology have enabled expansion of our ability to profile microbial diversity. These large datasets of taxonomic and functional diversity are key ...to better understanding microbial ecology. Machine learning has proven to be a useful approach for analyzing microbial community data and making predictions about outcomes including human and environmental health. Machine learning applied to microbial community profiles has been used to predict disease states in human health, environmental quality and presence of contamination in the environment, and as trace evidence in forensics. Machine learning has appeal as a powerful tool that can provide deep insights into microbial communities and identify patterns in microbial community data. However, often machine learning models can be used as black boxes to predict a specific outcome, with little understanding of how the models arrived at predictions. Complex machine learning algorithms often may value higher accuracy and performance at the sacrifice of interpretability. In order to leverage machine learning into more translational research related to the microbiome and strengthen our ability to extract meaningful biological information, it is important for models to be interpretable. Here we review current trends in machine learning applications in microbial ecology as well as some of the important challenges and opportunities for more broad application of machine learning to understanding microbial communities.
Verticillium wilt is one of the most severe diseases of olive. In this study, differential physiological and biochemical responses of two olive cultivars differing by their susceptibility to ...Verticillium dahliae were investigated using gene expression analyses and monitoring of some physiological and biochemical parameters. Results revealed that early up-regulation of SOD gene, together with a reduced scavenging system activity, resulted in higher H2O2 burst in the resistant cultivar as compared to the susceptible one. In addition, early and simultaneous up-regulation of chitinase and β-1,3-glucanase genes is correlated with reduced susceptibility and wilt symptoms in the resistant cultivar Sayali. The expression level of genes coding for PR10 protein, PLD and WRKY transcription factor was higher in the resistant cultivar, which indicates that induction of the SA pathway is essential for conferring resistance against V. dahliae and reduction of wilt symptoms. Furthermore, comparison of the SA-related genes expression patterns with those followed by the H2O2 burst, chitinase and β-1,3-glucanase genes, revealed that high SA level and H2O2 production, together with enhanced PR proteins activity are a key factor for resistance. As JA-related genes, a strong co-up-regulation of jasmonate ZIM domain and bHLH-binding factor responsive to JA was recorded in both cultivars, except that the extent of this induction was higher in the resistant cultivar. Similarly, the ERF related gene was differentially up-regulated in the two cultivars, which suggests that ET may play a role in the enhancement of olive resistance to V. dahliae. Overall, our findings provide important clues for designing strategies to restrict the effects of this disease.
•The expression of SA, JA, and ET related genes depends on the cultivar susceptibility level.•Early H2O2 burst and reduced scavenging system activity reduced root rot symptoms.•Simultaneous induction of chitinase and β-1,3-glucanase is associated with enhanced root resistance to V. dahliae.
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