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A significant consequence of climate change is the rising incidence of wildfires. When wildfires occur close to wine grape (Vitis vinifera) production areas, smoke-derived volatile phenolic compounds can be taken up by the grape berries, negatively affecting the flavor and aroma profile of the resulting wine and compromising the production value of entire vineyards. Evidence for the permeation of smoke-associated compounds into grape berries has been provided through metabolomics; however, the basis for grapevines' response to smoke at the gene expression level has not been investigated in detail. To address this knowledge gap, we employed time-course RNA sequencing to observe gene expression-level changes in grape berries in response to smoke exposure. Significant increases in gene expression (and enrichment of gene ontologies) associated with detoxification of reactive compounds, maintenance of redox homeostasis, and cell wall fortification were observed in response to smoke. These findings suggest that the accumulation of volatile phenols from smoke exposure activates mechanisms that render smoke-derived compounds less reactive while simultaneously fortifying intracellular defense mechanisms. The results of this work lend a better understanding of the molecular basis for grapevines' response to smoke and provide insight into the origins of smoke-taint-associated flavor and aroma attributes in wine produced from smoke-exposed grapes. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockades are actively adopted in diverse cancer types including metastatic melanoma and lung cancer. Despite of durable response in 20–30% of patients, we still lack molecular markers that could predict the patient responses reliably before treatment. Here we present a composite model for predicting anti-PD-1 response based on tumor mutation burden (TMB) and transcriptome sequencing data of 85 lung adenocarcinoma (LUAD) patients who received anti-PD-(L)1 treatment. We found that TMB was a good predictor (AUC = 0.81) for PD-L1 negative patients (n = 20). For PD-L1 positive patients (n = 65), we built an ensemble model of 100 XGBoost learning machines where gene expression, gene set activities and cell type composition were used as input features. The transcriptome-based models showed excellent accuracy (AUC > 0.9) and highlighted the contribution of T cell activities. Importantly, nonresponder patients with high prediction score turned out to have high CTLA4 expression, which suggested that neoadjuvant CTLA4 combination therapy might be effective for these patients. Our data and analysis results provide valuable insights into developing biomarkers and strategies for treating LUAD patients using immune checkpoint inhibitors. [ABSTRACT FROM AUTHOR]

The interplay between crystals and epithelial cells forms the cornerstone of kidney stone development, communication between epithelial cells and macrophages emerging as a pivotal role in this process. We conducted next-generation sequencing on the secreted exosomes of TCMK-1 cells treated with calcium oxalate monohydrate (OX_EXO) or controls (NC_EXO), and on the macrophage cell line RAW264.7 stimulated with OX_EXO or NC_EXO, followed by validation of differentially expressed target proteins and miRNAs through Western blot and PCR. UPSET plots were employed to identify genes co-targeted by exosomal miRNAs. Various bioinformatic analyses were employed to predict potential mechanisms of the dysregulated genes. We integrated sequencing data from the GEO database, and validated findings using clinical patient urine and kidney tissues. We identified 665 differentially expressed exosomal miRNAs between OX_EXO and NC_EXO. Among the top 10 down-regulated miRNAs, the most targeted genes were AAK1 and NUFIP2, whereas PLCB1 was significantly targeted among the top 10 up-regulated miRNAs. In clinical specimens, we confirmed the differential expressions of five homologous miRNAs, as well as CNOT3, CNCNA1C, APEX1, and TMEM199. In conclusion, treatment of TCMK-1 cells with calcium oxalate significantly alerted the expression profile of exosomal miRNAs, subsequently influencing gene expression in macrophages, thereby modulating the processes of kidney stone formation. [ABSTRACT FROM AUTHOR]

Obesity is characterized by dysregulated adipogenesis that leads to increased number and/or size of adipocytes. Understanding the molecular mechanisms governing adipogenesis is therefore key to designing therapeutic interventions against obesity. In our study, we analyzed 3'-end sequencing data that we generated from human preadipocytes and adipocytes, as well as previously published RNA-seq datasets, to elucidate mechanisms of regulation via long non-coding RNA (lncRNA), alternative splicing (AS) and alternative polyadenylation (APA). We discovered lncRNAs that have not been previously characterized but may be key regulators of white adipogenesis. We also detected 100 AS events and, using motif enrichment analysis, identified RNA binding proteins (RBPs) that could mediate exon skipping—the most prevalent AS event. In addition, we show that usage of alternative poly(A) sites in introns or 3'-UTRs of key adipogenesis genes leads to isoform diversity, which can have significant biological consequences on differentiation efficiency. We also identified RBPs that may modulate APA and defined how 3'-UTR APA can regulate gene expression through gain or loss of specific microRNA binding sites. Taken together, our bioinformatics-based analysis reveals potential therapeutic avenues for obesity through manipulation of lncRNA levels and the profile of mRNA isoforms via alternative splicing and polyadenylation. [ABSTRACT FROM AUTHOR]

Malaria transmission and endemicity in Africa remains hugely disproportionate compared to the rest of the world. The complex life cycle of P. falciparum (Pf) between the vertebrate human host and the anopheline vector results in differential expression of genes within and between hosts. An in-depth understanding of Pf interaction with various human genes through regulatory elements will pave way for identification of newer tools in the arsenal for malaria control. Therefore, the regulatory elements (REs) involved in the over- or under-expression of various host immune genes hold the key to elucidating alternative control measures that can be applied for disease surveillance, prompt diagnosis and treatment. We carried out an RNAseq analysis to identify differentially expressed genes and network elucidation of non-coding RNAs and target genes associated with immune response in individuals with different clinical outcomes. Raw RNAseq datasets, retrieved for analyses include individuals with severe (Gambia—20), symptomatic (Burkina Faso—15), asymptomatic (Mali—16) malaria as well as uninfected controls (Tanzania—20; Mali—36). Of the total 107 datasets retrieved, we identified 5534 differentially expressed genes (DEGs) among disease and control groups. A peculiar pattern of DEGs was observed, with individuals presenting with severe/symptomatic malaria having the highest and most diverse upregulated genes, while a reverse phenomenon was recorded among asymptomatic and uninfected individuals. In addition, we identified 141 differentially expressed micro RNA (miRNA), of which 78 and 63 were upregulated and downregulated respectively. Interactome analysis revealed a moderate interaction between DEGs and miRNAs. Of all identified miRNA, five were unique (hsa-mir-32, hsa-mir-25, hsa-mir-221, hsa-mir-29 and hsa-mir-148) because of their connectivity to several genes, including hsa-mir-221 connected to 16 genes. Six-hundred and eight differentially expressed long non coding RNA (lncRNA) were also identified, including SLC7A11, LINC01524 among the upregulated ones. Our study provides important insight into host immune genes undergoing differential expression under different malaria conditions. It also identified unique miRNAs and lncRNAs that modify and/or regulate the expression of various immune genes. These regulatory elements we surmise, have the potential to serve a diagnostic purpose in discriminating between individuals with severe/symptomatic malaria and those with asymptomatic infection or uninfected, following further clinical validation from field isolates. [ABSTRACT FROM AUTHOR]

Manipulation of the rumen microbial ecosystem in early life may affect ruminal fermentation and enhance the productive performance of dairy cows. The objective of this experiment was to evaluate the effects of dosing three different types of microbial inoculum on the rumen epithelium tissue (RE) transcriptome and the rumen epimural metatranscriptome (REM) in dairy calves. For this objective, 15 Holstein bull calves were enrolled in the study at birth and assigned to three different intraruminal inoculum treatments dosed orally once weekly from three to six weeks of age. The inoculum treatments were prepared from rumen contents collected from rumen fistulated lactating cows and were either autoclaved (control; ARF), processed by differential centrifugation to create the bacterial-enriched inoculum (BE), or through gravimetric separation to create the protozoal-enriched inoculum (PE). Calves were fed 2.5 L/d pasteurized waste milk 3x/d from 0 to 7 weeks of age and texturized starter until euthanasia at 9 weeks of age, when the RE tissues were collected for transcriptome and microbial metatranscriptome analyses, from four randomly selected calves from each treatment. The different types of inoculum altered the RE transcriptome and REM. Compared to ARF, 9 genes were upregulated in the RE of BE and 92 in PE, whereas between BE and PE there were 13 genes upregulated in BE and 114 in PE. Gene ontology analysis identified enriched GO terms in biological process category between PE and ARF, with no enrichment between BE and ARF. The RE functional signature showed different KEGG pathways related to BE and ARF, and no specific KEGG pathway for PE. We observed a lower alpha diversity index for RE microbiome in ARF (observed genera and Chao1 (p < 0.05)). Five microbial genera showed a significant correlation with the changes in host gene expression: Roseburia (25 genes), Entamoeba (two genes); Anaerosinus, Lachnospira, and Succiniclasticum were each related to one gene. sPLS-DA analysis showed that RE microbial communities differ among the treatments, although the taxonomic and functional microbial profiles show different distributions. Co-expression Differential Network Analysis indicated that both BE and PE had an impact on the abundance of KEGG modules related to acyl-CoA synthesis, type VI secretion, and methanogenesis, while PE had a significant impact on KEGGs related to ectoine biosynthesis and D-xylose transport. Our study indicated that artificial dosing with different microbial inocula in early life alters not only the RE transcriptome, but also affects the REM and its functions. [ABSTRACT FROM AUTHOR]

Marine microbial communities form the basis for the functioning of marine ecosystems and the conservation of biodiversity. With the application of metagenomics and metatranscriptomics in marine environmental studies, significant progress has been made in analysing the functioning of microbial communities as a whole. These molecular techniques are highly dependent on reliable, well-characterised, comprehensive and taxonomically diverse sequenced reference transcriptomes of microbial organisms. Here we present a set of 12 individual transcriptome assemblies derived from 6 representative diatom species from the northern Adriatic Sea grown under 2 environmentally relevant growth conditions (phosphate replete vs. phosphate deprived). After filtering the reads and assembly, an average number of 64,932 transcripts per assembly was obtained, of which an average of 8856 were assigned to functionally known proteins. Of all assigned transcripts, an average of 6483 proteins were taxonomically assigned to diatoms (Bacillariophyta). On average, a higher number of assigned proteins was detected in the transcriptome assemblies of diatoms grown under replete media condition. On average, 50% of the mapped proteins were shared between the two growth conditions. All recorded proteins in the dataset were classified into 24 COG categories, with approximately 25% belonging to the unknown function and the remaining 75% belonging to all other categories. The resulting diatom reference database for the northern Adriatic, focussing on the response to nutrient limitation as characteristic for the region and predicted for the future world oceans, provides a valuable resource for analysing environmental metatranscriptome and metagenome data. Each northern Adriatic transcriptome can also be used by itself as a reference database for the (meta)transcriptomes and gene expression studies of the associated species that will be generated in the future. [ABSTRACT FROM AUTHOR]

Genetic variations in the ovine ovulation rate, which are associated with the FecB mutation, provide useful models by which to explore the mechanisms regulating the development of mammalian antral follicles. In order to study the effects of the FecB mutation on cumulus cell differentiation, preovulatory follicles were aspirated and cumulus cells were isolated from three FecB genotypes (homozygous, heterozygous and wild type) of Small Tail Han (STH) sheep superstimulated with FSH. Transcriptome information from tens of thousands of cumulus cells was determined with the 10 × Genomics single-cell RNA-seq technology. Under the superovulation treatment, the observed number of preovulatory follicles in the ovaries of FecB carriers was still significantly higher than that in the wild-type (P < 0.05). The expression patterns of cumulus cells differed between FecB carriers and wild-type ewes. The screened cumulus cells could also be further divided into different cell clusters, and the differentiation states and fates of each group of cumulus cells also remained different, which supports the notion that heterogeneity in gene expression is prevalent in single cells. The oxidative phosphorylation pathway was significantly enriched in differentially expressed genes among the cell differentiation branch nodes of cumulus cells and among the differentially expressed genes of cumulus cells from the three genotypes. Combined with the important role of oxidative phosphorylation in the maturation of COCs, we suggest that the oxidative phosphorylation pathway of cumulus cells plays a crucial role in the differentiation process of cumulus cells and the mutation effect of the FecB gene. [ABSTRACT FROM AUTHOR]

Melia azedarach demonstrates strong salt tolerance and thrives in harsh saline soil conditions, but the underlying mechanisms are poorly understood. In this study, we analyzed gene expression under low, medium, and high salinity conditions to gain a deeper understanding of adaptation mechanisms of M. azedarach under salt stress. The GO (gene ontology) analysis unveiled a prominent trend: as salt stress intensified, a greater number of differentially expressed genes (DEGs) became enriched in categories related to metabolic processes, catalytic activities, and membrane components. Through the analysis of the category GO:0009651 (response to salt stress), we identified four key candidate genes (CBL7, SAPK10, EDL3, and AKT1) that play a pivotal role in salt stress responses. Furthermore, the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis revealed that DEGs were significantly enriched in the plant hormone signaling pathways and starch and sucrose metabolism under both medium and high salt exposure in comparison to low salt conditions. Notably, genes involved in JAZ and MYC2 in the jasmonic acid (JA) metabolic pathway were markedly upregulated in response to high salt stress. This study offers valuable insights into the molecular mechanisms underlying M. azedarach salt tolerance and identifies potential candidate genes for enhancing salt tolerance in M. azedarach. [ABSTRACT FROM AUTHOR]

Spatial transcriptomics (ST) assays represent a revolution in how the architecture of tissues is studied by allowing for the exploration of cells in their spatial context. A common element in the analysis is delineating tissue domains or "niches" followed by detecting differentially expressed genes to infer the biological identity of the tissue domains or cell types. However, many studies approach differential expression analysis by using statistical approaches often applied in the analysis of non-spatial scRNA data (e.g., two-sample t-tests, Wilcoxon's rank sum test), hence neglecting the spatial dependency observed in ST data. In this study, we show that applying linear mixed models with spatial correlation structures using spatial random effects effectively accounts for the spatial autocorrelation and reduces inflation of type-I error rate observed in non-spatial based differential expression testing. We also show that spatial linear models with an exponential correlation structure provide a better fit to the ST data as compared to non-spatial models, particularly for spatially resolved technologies that quantify expression at finer scales (i.e., single-cell resolution). [ABSTRACT FROM AUTHOR]

Necrotising enterocolitis (NEC) has a complex pathophysiology but the common end-point is ischaemia reperfusion injury (IRI) and intestinal necrosis. We have previously reported that RIC significantly reduces the intestinal injury in a rat model of NEC. Here we describe the changes in intestinal mRNA occurring in the intestine of animals exposed to IRI, both with and without RIC. Related rat-pups were randomly assigned to four groups: SHAM, IRI only, RIC only and RIC + IRI. IRI animals, underwent 40 min of intestinal ischaemia, and 90 min of reperfusion. Animals that underwent RIC had three cycles of 5 min of alternating ischaemia/reperfusion by means of a ligature applied to the hind limb. Samples from the terminal ileum were immediately stored in RNA-preserving media for later next generation sequencing and transciptome analysis using R v 3.6.1. Differential expression testing showed that 868 genes differentially expressed in animals exposed to RIC alone compared to SHAM and 135 in the IRI and RIC group compared to IRI alone. Comparison between these two sets showed that 25 genes were differentially expressed in both groups. Pro-inflammatory molecules: NF-ĸβ2, Cxcl1, SOD2 and Map3k8 all show reduced expression in response to RIC. Targeted gene analysis revealed increased expression in PI3K which is part of the so-called RISK-pathway which is a key part of the protective mechanisms of RIC in the heart. Overall, this transcriptomic analysis shows that RIC provides a protective effect to the intestine via anti-inflammatory pathways. This could be particularly relevant to treating and preventing NEC. [ABSTRACT FROM AUTHOR]

The progression of muscle development is a pivotal aspect of animal ontogenesis, where miRNA and mRNA exert substantial influence as prominent players. It is important to understand the molecular mechanisms involved in skeletal muscle development to enhance the quality and yield of meat produced by Leizhou goats. We employed RNA sequencing (RNA-SEQ) technology to generate miRNA-mRNA profiles in Leizhou goats, capturing their developmental progression at 0, 3, and 6 months of age. A total of 977 mRNAs and 174 miRNAs were found to be differentially expressed based on our analysis. Metabolic pathways, calcium signaling pathways, and amino acid synthesis and metabolism were found to be significantly enriched among the differentially expressed mRNA in the enrichment analysis. Meanwhile, we found that among these differentially expressed mRNA, some may be related to muscle development, such as MYL10, RYR3, and CSRP3. Additionally,, we identified five muscle-specific miRNAs (miR-127-3p, miR-133a-3p, miR-193b-3p, miR-365-3p, and miR-381) that consistently exhibited high expression levels across all three stages. These miRNAs work with their target genes (FHL3, SESN1, PACSIN3, LMCD1) to regulate muscle development. Taken together, our findings suggest that several miRNAs and mRNAs are involved in regulating muscle development and cell growth in goats. By uncovering the molecular mechanisms involved in muscle growth and development, these findings contribute valuable knowledge that can inform breeding strategies aimed at enhancing meat yield and quality in Leizhou goats. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is the leading cause of premature death. The death of cardiomyocytes (CMs) and the dysfunction of the remaining viable CMs are the main pathological factors contributing to heart failure (HF) following MI. This study aims to determine the transcriptional profile of CMs and investigate the heterogeneity among CMs under hypoxic conditions. Single-cell atlases of the heart in both the sham and MI groups were developed using single-cell data (GSE214611) downloaded from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). The heterogeneity among CMs was explored through various analyses including enrichment, pseudo time, and intercellular communication analysis. The marker gene of C5 was identified using differential expression analysis (DEA). Real-time polymerase chain reaction (RT-PCR), bulk RNA-sequencing dataset analysis, western blotting, immunohistochemical and immunofluorescence staining, Mito-Tracker staining, TUNEL staining, and flow cytometry analysis were conducted to validate the impact of the marker gene on mitochondrial function and cell apoptosis of CMs under hypoxic conditions. We identified a cell subcluster named C5 that exhibited a close association with mitochondrial malfunction and cellular apoptosis characteristics, and identified Slc25a4 as a significant biomarker of C5. Furthermore, our findings indicated that the expression of Slc25a4 was increased in failing hearts, and the downregulation of Slc25a4 improved mitochondrial function and reduced cell apoptosis. Our study significantly identified a distinct subcluster of CMs that exhibited strong associations with ventricular remodeling following MI. Slc25a4 served as the hub gene for C5, highlighting its significant potential as a novel therapeutic target for MI. [ABSTRACT FROM AUTHOR]

Phthorimaeaabsoluta is an invasive solanaceous plant pest with highly devastating effects on tomato plant. Heavy reliance on insecticide use to tackle the pest has been linked to insecticide resistance selection in P.absoluta populations. To underline insights on P.absoluta insecticide resistance mechanisms to diamides and avermectins, we evaluated the transcriptomic profile of parental (field-collected) and F8 (lab-reared) populations. Furthermore, to screen for the presence of organophosphate and pyrethroid resistance, we assessed the gene expression levels of acetylcholinesterase (ace1) and para-type voltage-gated sodium channel (VGSG) genes in the F1 to F8 lab-reared progeny of diamide and avermectin exposed P.absoluta field-collected populations. The VGSG gene showed up-regulation in 12.5% and down-regulation in 87.5% of the screened populations, while ace1 gene showed up-regulation in 37.5% and down-regulation in 62.5% of the screened populations. Gene ontology of the differentially expressed genes from both parental and eighth generations of diamide-sprayed P.absoluta populations revealed three genes involved in the metabolic detoxification of diamides in P.absoluta. Therefore, our study showed that the detoxification enzymes found could be responsible for P.absoluta diamide-based resistance, while behavioural resistance, which is stimulus-dependent, could be attributed to P.absoluta avermectin resistance. [ABSTRACT FROM AUTHOR]

New or mild heart failure (HF) is mainly caused by left ventricular dysfunction. We hypothesised that gene expression differ between the left (LV) and right ventricle (RV) and secondly by type of LV dysfunction. We compared gene expression through myocardial biopsies from LV and RV of patients undergoing elective coronary bypass surgery (CABG). Patients were categorised based on LV ejection fraction (EF), diastolic function and NT-proBNP into pEF (preserved; LVEF ≥ 45%), rEF (reduced; LVEF < 45%) or normal LV function. Principal component analysis of gene expression displayed two clusters corresponding to LV and RV. Up-regulated genes in LV included natriuretic peptides NPPA and NPPB, transcription factors/coactivators STAT4 and VGLL2, ion channel related HCN2 and LRRC38 associated with cardiac muscle contraction, cytoskeleton, and cellular component movement. Patients with pEF phenotype versus normal differed in gene expression predominantly in LV, supporting that diastolic dysfunction and structural changes reflect early LV disease in pEF. DKK2 was overexpressed in LV of HFpEF phenotype, potentially leading to lower expression levels of β-catenin, α-SMA (smooth muscle actin), and enhanced apoptosis, and could be a possible factor in the development of HFpEF. CXCL14 was down-regulated in both pEF and rEF, and may play a role to promote development of HF. [ABSTRACT FROM AUTHOR]

Numerous methods for bulk RNA sequence deconvolution have been developed to identify cellular targets of diseases by understanding the composition of cell types in disease-related tissues. However, issues of heterogeneity in gene expression between subjects and the shortage of reference single-cell RNA sequence data remain to achieve accurate bulk deconvolution. In our study, we investigated whether a new data generative method named sc-CMGAN and benchmarking generative methods (Copula, CTGAN and TVAE) could solve these issues and improve the bulk deconvolutions. We also evaluated the robustness of sc-CMGAN using three deconvolution methods and four public datasets. In almost all conditions, the generative methods contributed to improved deconvolution. Notably, sc-CMGAN outperformed the benchmarking methods and demonstrated higher robustness. This study is the first to examine the impact of data augmentation on bulk deconvolution. The new generative method, sc-CMGAN, is expected to become one of the powerful tools for the preprocessing of bulk deconvolution. [ABSTRACT FROM AUTHOR]

The oomycete Phytophthora palmivora infects the fruit of cacao trees (Theobroma cacao) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora, yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue-P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis-regulatory modules likely to be involved in cacao defense against P. palmivora. This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora-resistant cacao. [ABSTRACT FROM AUTHOR]

This study aims to use spatial transcriptomics to characterize the cell-type-specific expression profile associated with the microscopic features observed in Wooden Breast myopathy. 1 cm3 muscle sample was dissected from the cranial part of the right pectoralis major muscle from three randomly sampled broiler chickens at 23 days post-hatch and processed with Visium Spatial Gene Expression kits (10X Genomics), followed by high-resolution imaging and sequencing on the Illumina Nextseq 2000 system. WB classification was based on histopathologic features identified. Sequence reads were aligned to the chicken reference genome (Galgal6) and mapped to histological images. Unsupervised K-means clustering and Seurat integrative analysis differentiated histologic features and their specific gene expression pattern, including lipid laden macrophages (LLM), unaffected myofibers, myositis and vasculature. In particular, LLM exhibited reprogramming of lipid metabolism with up-regulated lipid transporters and genes in peroxisome proliferator-activated receptors pathway, possibly through P. Moreover, overexpression of fatty acid binding protein 5 could enhance fatty acid uptake in adjacent veins. In myositis regions, increased expression of cathepsins may play a role in muscle homeostasis and repair by mediating lysosomal activity and apoptosis. A better knowledge of different cell-type interactions at early stages of WB is essential in developing a comprehensive understanding. [ABSTRACT FROM AUTHOR]

Tanshinones, are a group of diterpenoid red pigments present in Danshen – an important herbal drug of Traditional Chinese Medicine which is a dried root of Salvia miltiorrhiza Bunge. Some of the tanshinones are sought after as pharmacologically active natural products. To date, the biosynthetic pathway of tanshinones has been only partially elucidated. These compounds are also present in some of the other Salvia species, i.a. from subgenus Perovskia, such as S. abrotanoides (Kar.) Sytsma and S. yangii B.T. Drew. Despite of the close genetic relationship between these species, significant qualitative differences in their diterpenoid profile have been discovered. In this work, we have used the Liquid Chromatography–Mass Spectrometry analysis to follow the content of diterpenoids during the vegetation season, which confirmed our previous observations of a diverse diterpenoid profile. As metabolic differences are reflected in different transcript profile of a species or tissues, we used metabolomics-guided transcriptomic approach to select candidate genes, which expression possibly led to observed chemical differences. Using an RNA-sequencing technology we have sequenced and de novo assembled transcriptomes of leaves and roots of S. abrotanoides and S. yangii. As a result, 134,443 transcripts were annotated by UniProt and 56,693 of them were assigned as Viridiplantae. In order to seek for differences, the differential expression analysis was performed, which revealed that 463, 362, 922 and 835 genes indicated changes in expression in four comparisons. GO enrichment analysis and KEGG functional analysis of selected DEGs were performed. The homology and expression of two gene families, associated with downstream steps of tanshinone and carnosic acid biosynthesis were studied, namely: cytochromes P-450 and 2-oxoglutarate-dependend dioxygenases. Additionally, BLAST analysis revealed existence of 39 different transcripts related to abietane diterpenoid biosynthesis in transcriptomes of S. abrotanoides and S. yangii. We have used quantitative real-time RT-PCR analysis of selected candidate genes, to follow their expression levels over the vegetative season. A hypothesis of an existence of a multifunctional CYP76AH89 in transcriptomes of S. abrotanoides and S. yangii is discussed and potential roles of other CYP450 homologs are speculated. By using the comparative transcriptomic approach, we have generated a dataset of candidate genes which provides a valuable resource for further elucidation of tanshinone biosynthesis. In a long run, our investigation may lead to optimization of diterpenoid profile in S. abrotanoides and S. yangii, which may become an alternative source of tanshinones for further research on their bioactivity and pharmacological therapy. [ABSTRACT FROM AUTHOR]

Mesangial proliferation is a diagnostic feature and a prognostic predictor of immunoglobulin A nephropathy (IgAN). We aimed to investigate the gene expression profiles of IgAN glomerulus according to the presence of mesangial proliferation. We performed spatial-specific transcriptomic profiling on kidney biopsy tissues using the GeoMx Digital Spatial Profiler. Twelve cases with three glomeruli for each case were profiled using direct pathologic classification (4 M1-IgAN, 4 M0-IgAN, and 4 donor controls). The results of enriched glom-specific genes demonstrated that M1-IgAN could be distinguished from controls (77 upregulated and 55 downregulated DEGs), while some DEGs were identified between M1-IgAN and M0-IgAN cases (24 upregulated and 8 downregulated DEGs) or between M0 and controls (1 upregulated and 16 downregulated DEGs). TCF21, an early podocyte damage marker, was the only differentially expressed gene (DEG) consistently upregulated in both M1-IgAN and M0-IgAN patients, whereas ATF3, EGR1, DUSP1, FOS, JUNB, KLF2, NR4A1, RHOB, and ZFP36 were consistently downregulated in IgAN cases. Glomeruli from M1-IgAN cases were significantly enriched for cell surface/adhesion molecules and gene expressions associated with vascular development or the extracellular matrix. Spatial transcriptomic analysis may contribute to dissecting structure-specific pathophysiology and molecular changes in IgAN. [ABSTRACT FROM AUTHOR]

Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey the steady state gene expression across the cell cycle and are inherently limited by their inability to resolve dynamic gene regulatory networks. Single cell RNAseq (scRNAseq) can identify different cell cycle transcriptomes if enough cycling cells are present, however some cells are not amenable to scRNAseq. Therefore, we merged two powerful strategies, the CDT1 and GMNN degrons used in Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cell cycle sensors and the ribosomal protein epitope tagging used in RiboTrap/Tag technologies to isolate cell cycle phase-specific mRNA for sequencing. The resulting cell cycle dependent, tagged ribosomal proteins (ccTaggedRP) were differentially expressed during the cell cycle, had similar subcellular locations as endogenous ribosomal proteins, incorporated into ribosomes and polysomes, and facilitated the recovery of cell cycle phase-specific RNA for sequencing. ccTaggedRP has broad applications to investigate phase-specific gene expression in complex cell populations. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the molecular mechanism of sporotrichosis and identify possible novel therapeutic targets. Total RNA was extracted from skin lesion samples from sporotrichosis patients and used to construct a long-chain RNA transcriptome library and miRNA transcriptome library for whole transcriptome sequencing. The differentially expressed genes (DEGs) between the groups were identified, and then Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis enrichment analyses were performed based on the DEGs. An lncRNA-miRNA-mRNA ceRNA network was constructed. The expressions of JAK/STAT pathway-related proteins were detected in the patient and control tissues using RT-qPCR and Western blot analysis. Enrichment analysis showed that the DEGs were mainly enriched in various infectious diseases and immune response-related signaling pathways. Competing endogenous RNA network analysis was performed and identified the hub lncRNAs, miRNAs, and mRNAs. Compared with the control group, the mRNA expressions of SOCS3, IL-6, and JAK3 were significantly upregulated, while the expression of STAT3 did not change significantly. Also, the protein expressions of SOCS3, IL-6, JAK3, and STAT3, as well as phosphorylated JAK3 and STAT3, were significantly upregulated. We identified 671 lncRNA DEGs, 3281 mRNA DEGs, and 214 miRNA DEGs to be involved in Sporothrix globosa infection. The study findings suggest that the JAK/STAT pathway may be a therapeutic target for sporotrichosis. [ABSTRACT FROM AUTHOR]

Prodigiosin, a red pigment produced by Hahella chejuensis, a marine-derived microorganism, has several biological functions, including antimicrobial activity and inflammatory relief. In this study, the antibacterial activity of prodigiosin against skin microorganisms was explored. Paper disc assay on skin bacterial cells revealed that Cutibacterium acnes related to acne vulgaris highly susceptible to prodigiosin. MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) were determined on Cutibacterium species. The RNA-seq analysis of prodigiosin-treated C. acnes cells was performed to understand the antibacterial mechanism of prodigiosin. Among changes in the expression of hundreds of genes, the expression of a stress-responsive sigma factor encoded by sigB increased. Conversely, the gene expression of cell wall biosynthesis and energy metabolism was inhibited by prodigiosin. Specifically, the expression of genes related to the metabolism of porphyrin, a pro-inflammatory metabolite, was significantly reduced. Therefore, prodigiosin could be used to control C. acnes. Our study provided new insights into the antimicrobial mechanism of prodigiosin against C. acnes strains. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy is a common complication of long-term diabetes and that could lead to vision loss. Unfortunately, early diabetic retinopathy remains poorly understood. There is no effective way to prevent or treat early diabetic retinopathy until patients develop later stages of diabetic retinopathy. Elevated acellular capillary density is considered a reliable quantitative trait present in the early development of retinopathy. Hence, in this study, we interrogated whole retinal vascular transcriptomic changes via a Nile rat model to better understand the early pathogenesis of diabetic retinopathy. We uncovered the complexity of associations between acellular capillary density and the joint factors of blood glucose, diet, and sex, which was modeled through a Bayesian network. Using segmented regressions, we have identified different gene expression patterns and enriched Gene Ontology (GO) terms associated with acellular capillary density increasing. We developed a random forest regression model based on expression patterns of 14 genes to predict the acellular capillary density. Since acellular capillary density is a reliable quantitative trait in early diabetic retinopathy, and thus our model can be used as a transcriptomic clock to measure the severity of the progression of early retinopathy. We also identified NVP-TAE684, geldanamycin, and NVP-AUY922 as the top three potential drugs which can potentially attenuate the early DR. Although we need more in vivo studies in the future to support our re-purposed drugs, we have provided a data-driven approach to drug discovery. [ABSTRACT FROM AUTHOR]

Cancer-associated fibroblasts (CAFs), a prominent component of the tumor microenvironment, play an important role in tumor development, invasion, and drug resistance. The expression of distinct "CAF-markers" which separates CAFs from normal fibroblasts and epithelial cells, have traditionally been used to identify them. These commonly used CAF-markers have been reported to differ greatly across different CAF subpopulations, even within a cancer type. Using an unbiased -omic approach from public data and in-house RNAseq data from patient derived novel CAF cells, TIMP-1, SPARC, COL1A2, COL3A1 and COL1A1 were identified as potential CAF-markers by differential gene expression analysis using publicly available single cell sequencing data and in-house RNAseq data to distinguish CAF populations from tumor epithelia and normal oral fibroblasts. Experimental validation using qPCR and immunofluorescence revealed CAF-specific higher expression of TIMP-1 and COL1A2 as compared to other markers in 5 novel CAF cells, derived from patients of diverse gender, habits and different locations of head and neck squamous cell carcinoma (HNSC). Upon immunohistochemical (IHC) analysis of FFPE blocks however, COL1A2 showed better differential staining between tumor epithelia and tumor stroma. Similar data science driven approach utilizing single cell sequencing and RNAseq data from stabilized CAFs can be employed to identify CAF-markers in various cancers. [ABSTRACT FROM AUTHOR]

This study compared the expression of TP53 in lymphocytes from malignant melanoma (MM) patients with positive sentinel nodes to healthy controls (HCs) following exposure to various doses of UVA radiation. The Lymphocyte Genome Sensitivity (LGS) assay indicated significant differences in DNA damage in lymphocytes between MM patients and HCs. qPCR data demonstrated an overall 3.4-fold increase in TP53 expression in lymphocytes from MM patients compared to healthy controls, following treatment with 0.5 mW/cm2 UVA radiation. Western blotting confirmed that p53 expression was increased in MM lymphocytes following UVA exposure compared to healthy individuals. Genome transcriptome profiling data displayed differences in gene expression between UVA-treated lymphocytes from MM patients and HCs. Peripheral lymphocytes from MM patients are more susceptible to the genotoxic effects of UVA compared to healthy individuals. Our previous studies showed that UVA exposure of various intensities caused significant differences in the levels of DNA damage between lymphocytes from cancer patients compared to HCs through the LGS assay. The present study's results provide further credibility to the LGS assay as a screening test for cancer detection. Peripheral lymphocytes could be a promising blood biopsy biomarker for staging of carcinomas and prevention of carcinoma progression at early stages. [ABSTRACT FROM AUTHOR]

Hyperlipidemia, a common metabolic disease, is a risk factor for cardiovascular diseases, Poria cocos (PC) and Alismatis rhizoma (AR) serve as a potential treatment. A systematic approach based on transcriptome sequencing analysis and bioinformatics methods was developed to explore the synergistic effects of PC–AR and identify major compounds and potential targets. The phenotypic characteristics results indicated that the high dose (4.54 g/kg) of PC–AR reduced total cholesterol (TC), elevated high-density lipoprotein cholesterol (HDL-C) levels, and improved hepatocyte morphology, as assessed via hematoxylin and eosin (H&E) staining. Transcriptomic profiling processing results combined with GO enrichment analysis to identify the overlapping genes were associated with inflammatory responses. The cytokine-cytokine receptor interaction pathway was found as a potential key pathway using geneset enrichment analysis. Core enrichment targets were selected according to the PC–AR's fold change versus the model. Real-time quantitative PCR analysis validated that PC–AR significantly downregulated the expression of Cxcl10, Ccl2, Ccl4, Cd40 and Il-1β mRNA (P < 0.05). Molecular docking analysis revealed the significant compounds of PC–AR and the potential binding patterns of the critical compounds and targets. This study provides further evidence that the therapeutic effects of PC–AR on hyperlipidemia in rats through the regulation of inflammation-related targets. [ABSTRACT FROM AUTHOR]

Understanding host-microbial interactions in the rumen and its influence on desirable production traits may lead to potential microbiota manipulation or genetic selection for improved cattle feed efficiency. This study investigated the host transcriptome and its correlation with the rumen archaea and bacteria differential abundance of two pure beef cattle breeds (Angus and Charolais) and one composite beef hybrid (Kinsella) divergent for residual feed intake (RFI; low-RFI vs. high-RFI). Using RNA-Sequencing of rumen tissue and 16S rRNA gene amplicon sequencing, differentially expressed genes (FDR ≤ 0.05, |log2(Fold-change) >|2) and differentially abundant (p-value < 0.05) archaea and bacteria amplicon sequence variants (ASV) were determined. Significant correlations between gene expression and ASVs (p-value < 0.05) were determine using Spearman correlation. Interesting associations with muscle contraction and the modulation of the immune system were observed for the genes correlated with bacterial ASVs. Potential functional candidate genes for feed efficiency status were identified for Angus (CCL17, CCR3, and CXCL10), Charolais (KCNK9, GGT1 and IL6), and Kinsella breed (ESR2). The results obtained here provide more insights regarding the applicability of target host and rumen microbial traits for the selection and breeding of more feed efficient beef cattle. [ABSTRACT FROM AUTHOR]

The purpose of this study was to explore the role of coixendide (Coix) combine with temozolomide (TMZ) in the treatment of Glioblastoma (GBM) and explore its possible mechanism. CCK-8 was used to determine the inhibitory rate of Coix group, TMZ group and drug combination group on GBM cells, and the combination index (CI) was calculated to determine whether they had synergistic effect. Then RNA was extracted from each group, transcriptome sequencing was performed, and differentially expressed genes (DEGs) were identified. The possible mechanism was analyzed by GO enrichment analysis and KEGG enrichment analysis. The CI of Coix and TMZ indicating a synergistic effect when TMZ concentration is 0.1 mg/ml and Coix concentration is 2 mg/ml. Transcriptome sequencing analysis showed that interferon (IFN) related genes were down-regulated by Coix and up-regulated by TMZ and combined drugs, however, the up-regulation induced by combined drugs was less than that of TMZ. Besides IFN related genes, cholesterol metabolism pathway were also been regulated. Coix and TMZ have synergistic effects in the treatment of GBM at certain doses. RNA-Seq results suggested that the abnormal on genetic materials caused by DNA damage induced by TMZ treatment can be sensed by IFN related genes and activates antiviral IFN signaling, causing the activation of repairing mechanism and drug resistance. Coix inhibits IFN related genes, thereby inhibits drug resistance of TMZ. In addition, the activation of ferroptosis and the regulation of DEGs in cholesterol metabolism pathway were also contributed to the synergistic effects of Coix and TMZ. [ABSTRACT FROM AUTHOR]

Single cell RNA sequencing has a central role in immune profiling, identifying specific immune cells as disease markers and suggesting therapeutic target genes of immune cells. Immune cell-type annotation from single cell transcriptomics is in high demand for dissecting complex immune signatures from multicellular blood and organ samples. However, accurate cell type assignment from single-cell RNA sequencing data alone is complicated by a high level of gene expression heterogeneity. Many computational methods have been developed to respond to this challenge, but immune cell annotation accuracy is not highly desirable. We present ImmunIC, a simple and robust tool for immune cell identification and classification by combining marker genes with a machine learning method. With over two million immune cells and half-million non-immune cells from 66 single cell RNA sequencing studies, ImmunIC shows 98% accuracy in the identification of immune cells. ImmunIC outperforms existing immune cell classifiers, categorizing into ten immune cell types with 92% accuracy. We determine peripheral blood mononuclear cell compositions of severe COVID-19 cases and healthy controls using previously published single cell transcriptomic data, permitting the identification of immune cell-type specific differential pathways. Our publicly available tool can maximize the utility of single cell RNA profiling by functioning as a stand-alone bioinformatic cell sorter, advancing cell-type specific immune profiling for the discovery of disease-specific immune signatures and therapeutic targets. [ABSTRACT FROM AUTHOR]

Recurring heat and drought episodes present challenges to the sustainability of grape production worldwide. We investigated the impacts of heat and drought stress on transcriptomic and metabolic responses of berries from two wine grape varieties. Cabernet Sauvignon and Riesling grapevines were subjected to one of four treatments during early fruit ripening: (1) drought stress only, (2) heat stress only, (3) simultaneous drought and heat stress, (4) no drought or heat stress (control). Berry metabolites, especially organic acids, were analyzed, and time-course transcriptome analysis was performed on samples before, during, and after the stress episode. Both alone and in conjunction with water stress, heat stress had a much more significant impact on berry organic acid content, pH, and titratable acidity than water stress. This observation contrasts with previous reports for leaves, which responded more strongly to water stress, indicating that grape berries display a distinct, organ-specific response to environmental stresses. Consistent with the metabolic changes, the global transcriptomic analysis revealed that heat stress had a more significant impact on gene expression in grape berries than water stress in both varieties. The differentially expressed genes were those associated with the tricarboxylic acid cycle and glyoxylate cycle, mitochondrial electron transport and alternative respiration, glycolysis and gluconeogenesis, carbohydrate allocation, ascorbate metabolism, and abiotic stress signaling pathways. Knowledge regarding how environmental stresses, alone and in combination, impact the berry metabolism of different grape varieties will form the basis for developing recommendations for climate change mitigation strategies and genetic improvement. [ABSTRACT FROM AUTHOR]

Angelica dahurica (Fisch. ex Hoffm.) Benth.et Hook.f.var.formosana (Boiss.) Shan et Yuan (A. dahurica) is a well-known medicinal plant that has a wide range of applications in the pharmaceutical, food, cosmetic, and other industries. However, the issue of early bolting has emerged as a major hindrance to its production. This problem not only reduces the yield of A. dahurica, but also has an impact on its active ingredients. To date, the molecular factors that contribute to early bolting and its impact on the growth of A. dahurica have not been thoroughly investigated. Therefore, we conducted a transcriptome study using the Illumina NovaSeq 6000 on two developmental types: early-bolting and non-bolting (normal) roots of A. dahurica. We obtained 2,185 up-regulated and 1,414 down-regulated genes in total. Many of the identified transcripts were related to genes involved in early bolting. The gene ontology analysis revealed several differentially expressed genes that are crucial in various pathways, primarily associated with cellular, molecular, and biological processes. Additionally, the morphological characteristics and coumarin content in the early bolting roots of A. dahurica were significantly altered. This study provides insight into the transcriptomic regulation of early bolting in A. dahurica, which can potentially be utilized to enhance its medicinal properties. [ABSTRACT FROM AUTHOR]

Tea plants are crops with economic, health and cultural value. Catechin, caffeine and theanine are the main secondary metabolites of taste. In the process of germplasm collection, we found a resource in the Sandu Aquatic Autonomous County of Guizhou (SDT) that possessed significantly different characteristic metabolites compared with the cultivar 'Qiancha 1'. SDT is rich in theobromine and theophylline, possesses low levels of (−)-epicatechin-3-gallate, (−)-epigallocatechin-3-gallate, and theanine content, and is almost free of caffeine. However, research on this tea resource is limited. Full-length transcriptome analysis was performed to investigate the transcriptome and gene expression of these metabolites. In total, 78,809 unique transcripts were obtained, of which 65,263 were complete coding sequences. RNA-seq revealed 3415 differentially expressed transcripts in the tender leaves of 'Qiancha 1' and 'SDT'. Furthermore, 2665, 6231, and 2687 differentially expressed transcripts were found in different SDT tissues. These differentially expressed transcripts were enriched in flavonoid and amino acid metabolism processes. Co-expression network analysis identified five modules associated with metabolites and found that genes of caffeine synthase (TCS) may be responsible for the low caffeine content in SDT. Phenylalanine ammonia lyase (PAL), glutamine synthetase (GS), glutamate synthase (GOGAT), and arginine decarboxylase (ADC) play important roles in the synthesis of catechin and theanine. In addition, we identified that ethylene resposive factor (ERF) and WRKY transcription factors may be involved in theanine biosynthesis. Overall, our study provides candidate genes to improve understanding of the synthesis mechanisms of these metabolites and provides a basis for molecular breeding of tea plant. [ABSTRACT FROM AUTHOR]

The impact of climate change on biodiversity has stimulated the need to understand environmental stress responses, particularly for ecosystem engineers whose responses to climate affect large numbers of associated organisms. Distinct species differ substantially in their resilience to thermal stress but there are also within-species variations in thermal tolerance for which the molecular mechanisms underpinning such variation remain largely unclear. Intertidal mussels are well-known for their role as ecosystem engineers. First, we exposed two genetic lineages of the intertidal mussel Perna perna to heat stress treatments in air and water. Next, we ran a high throughput RNA sequencing experiment to identify differences in gene expression between the thermally resilient eastern lineage and the thermally sensitive western lineage. We highlight different thermal tolerances that concord with their distributional ranges. Critically, we also identified lineage-specific patterns of gene expression under heat stress and revealed intraspecific differences in the underlying transcriptional pathways in response to warmer temperatures that are potentially linked to the within-species differences in thermal tolerance. Beyond the species, we show how unravelling within-species variability in mechanistic responses to heat stress promotes a better understanding of global evolutionary trajectories of the species as a whole in response to changing climate. [ABSTRACT FROM AUTHOR]

Zebrafish have the capacity to fully regenerate the heart after an injury, which lies in sharp contrast to the irreversible loss of cardiomyocytes after a myocardial infarction in humans. Transcriptomics analysis has contributed to dissect underlying signaling pathways and gene regulatory networks in the zebrafish heart regeneration process. This process has been studied in response to different types of injuries namely: ventricular resection, ventricular cryoinjury, and genetic ablation of cardiomyocytes. However, there exists no database to compare injury specific and core cardiac regeneration responses. Here, we present a meta-analysis of transcriptomic data of regenerating zebrafish hearts in response to these three injury models at 7 days post injury (7dpi). We reanalyzed 36 samples and analyzed the differentially expressed genes (DEG) followed by downstream Gene Ontology Biological Processes (GO:BP) analysis. We found that the three injury models share a common core of DEG encompassing genes involved in cell proliferation, the Wnt signaling pathway and genes that are enriched in fibroblasts. We also found injury-specific gene signatures for resection and genetic ablation, and to a lower extent the cryoinjury model. Finally, we present our data in a user-friendly web interface that displays gene expression signatures across different injury types and highlights the importance to consider injury-specific gene regulatory networks when interpreting the results related to cardiac regeneration in the zebrafish. The analysis is freely available at: https://mybinder.org/v2/gh/MercaderLabAnatomy/PUB_Botos_et_al_2022_shinyapp_binder/HEAD?urlpath=shiny/bus-dashboard/. [ABSTRACT FROM AUTHOR]

Hirudin is a pharmacologically active substance in leeches with potent blood anticoagulation properties. Although recombinant hirudin production isolated from Hirudo medicinalis Linnaeus and Hirudinaria manillensis Lesson is known, to our knowledge, this study is the first to report recombinant hirudin expression and production from Hirudo nipponia Whitman. Thus, the present study aimed to clone and characterize the full-length cDNA of a candidate hirudin gene (c16237_g1), which is localized on the salivary gland transcriptome of H. nipponia, and further evaluate its recombinant production using a eukaryotic expression system. The 489-bp cDNA possessed several properties of the hirudin "core" motifs associated with binding to the thrombin catalytic pocket. A fusion expression vector (pPIC9K-hirudin) was constructed and successfully transformed into Pichia pastoris strain GS115 via electroporation. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blot analysis confirmed hirudin expression. The recombinant protein was expressed with a yield of 6.68 mg/L culture. Mass spectrometry analysis further confirmed target protein expression. The concentration and antithrombin activity of purified hirudin were 1.67 mg/mL and 14,000 ATU/mL, respectively. These findings provide a basis for further elucidating the molecular anticoagulation mechanism of hirudin, and address China's growing market demand for engineered H. nipponia-derived hirudin and hirudin-based drugs. [ABSTRACT FROM AUTHOR]

Flesh color is an important quality of melon (Cucumis melo L.) and is determined mainly by carotenoid content, awarding them with colors, aromas, and nutrients. enhancing the nutritional and health benefits of fruits and vegetables for humans. In this study, we performed transcriptomic analysis of two melon inbred line "B-14" (orange-flesh) and "B-6" (white-flesh) at three developmental stages. We observed that the β-carotene content of inbred line "B-6" (14.232 μg/g) was significantly lower than that of inbred line "B-14" (0.534 μg/g). RNA-sequencing and quantitative reverse transcription PCR analyses were performed to identify differentially expressed genes (DEGs) between the two inbred lines at different stages; the DEGs were analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes databases (KEGG). We identified 33 structural DEGs in different developmental periods of the two lines that were related to carotenoid metabolism. Among them, PSY, Z-ISO, ZDS, CRTISO, CCD4, VDE1, and NCED2 were highly correlated with carotenoid content. Thus, this study provides a basis for molecular mechanism of carotenoid biosynthesis and flesh color in melon fruit. [ABSTRACT FROM AUTHOR]

We evaluated the effect of acute hypobaric hypoxia (AHH) on the hippocampal region of the brain in early-stage spontaneously hypertensive male rats. The rats were classified into a control (ground level; ~ 400 m altitude) group and an AHH experimental group placed in an animal hypobaric chamber at a simulated altitude of 5500 m for 24 h. RNA-Seq analysis of the brains and hippocampi showed that differentially expressed genes (DEGs) were primarily associated with ossification, fibrillar collagen trimer, and platelet-derived growth factor binding. The DEGs were classified into functional categories including general function prediction, translation, ribosomal structure and biogenesis, replication, recombination, and repair. Pathway enrichment analysis revealed that the DEGs were primarily associated with relaxin signaling, PI3K-Akt signaling, and amoebiasis pathways. Protein–protein interaction network analysis indicated that 48 DEGs were involved in both inflammation and energy metabolism. Further, we performed validation experiments to show that nine DEGs were closely associated with inflammation and energy metabolism, of which two (Vegfa and Angpt2) and seven (Acta2, Nfkbia, Col1a1, Edn1, Itga1, Ngfr, and Sgk1) genes showed up and downregulated expression, respectively. Collectively, these results indicated that inflammation and energy metabolism-associated gene expression in the hippocampus was altered in early-stage hypertension upon AHH exposure. [ABSTRACT FROM AUTHOR]

Transcriptome profiling is a mainstay of translational cancer research and is increasingly finding its way into precision oncology. While bulk RNA sequencing (RNA-seq) is widely available, high investment costs and long data return time are limiting factors for clinical applications. We investigated a portable nanopore long-read sequencing device (MinION, Oxford Nanopore Technologies) for transcriptome profiling of tumors. In particular, we investigated the impact of lower coverage than that of larger sequencing devices by comparing shallow nanopore RNA-seq data with short-read RNA-seq data generated using reversible dye terminator technology (Illumina) for ten samples representing four cancer types. Coupled with ShaNTi (Shallow Nanopore sequencing for Transcriptomics), a newly developed data processing pipeline, a turnaround time of five days was achieved. The correlation of normalized gene-level counts between nanopore and Illumina RNA-seq was high for MinION but not for very low-throughput Flongle flow cells (r = 0.89 and r = 0.24, respectively). A cost-saving approach based on multiplexing of four samples per MinION flow cell maintained a high correlation with Illumina data (r = 0.56–0.86). In addition, we compared the utility of nanopore and Illumina RNA-seq data for analysis tools commonly applied in translational oncology: (1) Shallow nanopore and Illumina RNA-seq were equally useful for inferring signaling pathway activities with PROGENy. (2) Highly expressed genes encoding kinases targeted by clinically approved small-molecule inhibitors were reliably identified by shallow nanopore RNA-seq. (3) In tumor microenvironment composition analysis, quanTIseq performed better than CIBERSORT, likely due to higher average expression of the gene set used for deconvolution. (4) Shallow nanopore RNA-seq was successfully applied to detect fusion genes using the JAFFAL pipeline. These findings suggest that shallow nanopore RNA-seq enables rapid and biologically meaningful transcriptome profiling of tumors, and warrants further exploration in precision cancer medicine studies. [ABSTRACT FROM AUTHOR]

Lettuce (Lactuca sativa) is grown in various parts of the world for use as a leafy vegetable. Although the use of light-emitting diode (LED) in controlled plant production systems has been successfully used to enhance nutritional quality and plant growth efficiently, the molecular basis of lettuce's response to varying light spectra is not studied. Using next-generation sequencing, we have analyzed the transcriptomes of leaf lettuce (Lactuca sativa var. 'New Red Fire') grown hydroponically in a modular agricultural production system under three different types of LED lighting: red, blue, and white light. Illumina HiSeq sequencing platform was used to generate paired-end sequence reads (58 Gb raw and 54 Gb clean data) of the transcriptome of lettuce leaves exposed to varying light spectra. The de novo assembled final transcriptome contained 74,096 transcripts. Around 53% and 39% of the assembled transcripts matched to the UniProt and RefSeq RNA sequences, respectively. The validation of the differentially expressed transcripts using RT-qPCR showed complete agreement with RNA-Seq data for 27 transcripts. A comparison of the blue versus red light treatments showed the highest number of significantly differentially expressed transcripts. Among the transcripts significantly up-regulated in blue-light-exposed leaves compared to white-light-exposed leaves, ~ 26% were involved in the 'response to stress'. Among the transcripts significantly upregulated under red light compared to white light, ~ 6% were associated with 'nucleosome assembly' and other processes, such as 'oxidation–reduction process' and 'response to water deprivation' were significantly enriched. Thus, the result from the current study provides deeper insights into differential gene expression patterns and associated functional aspects under varying light qualities. [ABSTRACT FROM AUTHOR]

Widespread, debilitating and often treatment-resistant, depression and other stress-related neuropsychiatric disorders represent an urgent unmet biomedical and societal problem. Although animal models of these disorders are commonly used to study stress pathogenesis, they are often difficult to translate across species into valuable and meaningful clinically relevant data. To address this problem, here we utilized several cross-species/cross-taxon approaches to identify potential evolutionarily conserved differentially expressed genes and their sets. We also assessed enrichment of these genes for transcription factors DNA-binding sites down- and up- stream from their genetic sequences. For this, we compared our own RNA-seq brain transcriptomic data obtained from chronically stressed rats and zebrafish with publicly available human transcriptomic data for patients with major depression and their respective healthy control groups. Utilizing these data from the three species, we next analyzed their differential gene expression, gene set enrichment and protein–protein interaction networks, combined with validated tools for data pooling. This approach allowed us to identify several key brain proteins (GRIA1, DLG1, CDH1, THRB, PLCG2, NGEF, IKZF1 and FEZF2) as promising, evolutionarily conserved and shared affective 'hub' protein targets, as well as to propose a novel gene set that may be used to further study affective pathogenesis. Overall, these approaches may advance cross-species brain transcriptomic analyses, and call for further cross-species studies into putative shared molecular mechanisms of affective pathogenesis. [ABSTRACT FROM AUTHOR]

Retinopathies are multifactorial diseases with complex pathologies that eventually lead to vision loss. Animal models facilitate the understanding of the pathophysiology and identification of novel treatment options. However, each animal model reflects only specific disease aspects and understanding of the specific molecular changes in most disease models is limited. Here, we conducted transcriptome analysis of murine ocular tissue transduced with recombinant Adeno-associated viruses (AAVs) expressing either human VEGF-A, TNF-α, or IL-6. VEGF expression led to a distinct regulation of extracellular matrix (ECM)-associated genes. In contrast, both TNF-α and IL-6 led to more comparable gene expression changes in interleukin signaling, and the complement cascade, with TNF-α-induced changes being more pronounced. Furthermore, integration of single cell RNA-Sequencing data suggested an increase of endothelial cell-specific marker genes by VEGF, while TNF-α expression increased the expression T-cell markers. Both TNF-α and IL-6 expression led to an increase in macrophage markers. Finally, transcriptomic changes in AAV-VEGF treated mice largely overlapped with gene expression changes observed in the oxygen-induced retinopathy model, especially regarding ECM components and endothelial cell-specific gene expression. Altogether, our study represents a valuable investigation of gene expression changes induced by VEGF, TNF-α, and IL-6 and will aid researchers in selecting appropriate animal models for retinopathies based on their agreement with the human pathophysiology. [ABSTRACT FROM AUTHOR]

Osmanthus fragrans is an important evergreen species with both medicinal and ornamental value in China. Given the low efficiency of callus proliferation and the difficulty of adventitious bud differentiation, tissue culture and regeneration systems have not been successfully established for this species. To understand the mechanism of callus proliferation, transcriptome sequencing and endogenous hormone content determination were performed from the initial growth stages to the early stages of senescence on O. fragrans calli. In total, 47,340 genes were identified by transcriptome sequencing, including 1798 previously unidentified genes specifically involved in callus development. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) was significantly enriched in plant hormone signal transduction pathways. Furthermore, our results from the orthogonal projections to latent structures discrimination analysis (OPLS-DA) of six typical hormones in five development stages of O. fragrans calli showed jasmonic acid (JA) could play important role in the initial stages of calli growth, whereas JA and auxin (IAA) were dominant in the early stages of calli senescence. Based on the weighted gene co-expression network analysis, OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b were selected as hub genes from the modules with the significant relevance to JA and IAA respectively. The gene regulation network and quantitative real-time PCR implied that during the initial stages of callus growth, the transcription factors (TFs) OfERF4 and OfMYC2a could down-regulate the expression of hub genes OfSRC2 and OfPP2CD5, resulting in decreased JA content and rapid callus growth; during the late stage of callus growth, the TFs OfERF4, OfMYC2a and OfTGA21c, OfHSFA1 could positively regulate the expression of hub genes OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b, respectively, leading to increased JA and IAA contents and inducing the senescence of O. fragrans calli. Hopefully, our results could provide new insights into the molecular mechanism of the proliferation of O. fragrans calli. [ABSTRACT FROM AUTHOR]

In plants, the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) perceives UV-B and induces UV-B responses. UVR8 absorbs a range of UV-B (260–335 nm). However, the responsiveness of plants to each UV-B wavelength has not been intensively studied so far. Here, we performed transcriptome and metabolome analyses of Arabidopsis using UV light emitting diodes (LEDs) with peak wavelengths of 280 and 310 nm to investigate the differences in the wavelength-specific UV-B responses. Irradiation with both UV-LEDs induced gene expression of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), which has a central role in the UVR8 signaling pathway. However, the overall transcriptomic and metabolic responses to 280 and 310 nm UV-LED irradiation were different. Most of the known UV-B-responsive genes, such as defense-related genes, responded only to 280 nm UV-LED irradiation. Lipids, polyamines and organic acids were the metabolites most affected by 280 nm UV-LED irradiation, whereas the effect of 310 nm UV-LED irradiation on the metabolome was considerably less. Enzymatic genes involved in the phenylpropanoid pathway upstream in anthocyanin biosynthesis were up-regulated only by 280 nm UV-LED irradiation. These results revealed that the responsivenesses of Arabidopsis to 280 and 310 nm UV-B were significantly different, suggesting that UV-B signaling is mediated by more complex pathways than the current model. [ABSTRACT FROM AUTHOR]

Transcriptional analysis of the network of transcription regulators and target pathways in exposed organisms may be a hard task when their genome remains unknown. The development of hundreds of qPCR assays, including primer design and normalization of the results with the appropriate housekeeping genes, seems an unreachable task. Alternatively, we took advantage of a whole transcriptome study on Rhinella arenarum larvae exposed to the organophosphorus pesticides azinphos-methyl and chlorpyrifos to evaluate the transcriptional effects on a priori selected groups of genes. This approach allowed us to evaluate the effects on hypothesis-selected pathways such as target esterases, detoxifying enzymes, polyamine metabolism and signaling, and regulatory pathways modulating them. We could then compare the responses at the transcriptional level with previously described effects at the enzymatic or metabolic levels to obtain global insight into toxicity–response mechanisms. The effects of both pesticides on the transcript levels of these pathways could be considered moderate, while chlorpyrifos-induced responses were more potent and earlier than those elicited by azinphos-methyl. Finally, we inferred a prevailing downregulation effect of pesticides on signaling pathways and transcription factor transcripts encoding products that modulate/control the polyamine and antioxidant response pathways. We also tested and selected potential housekeeping genes based on those reported for other species. These results allow us to conduct future confirmatory studies on pesticide modulation of gene expression in toad larvae. [ABSTRACT FROM AUTHOR]

Holometabolous insects are predominantly motionless during metamorphosis, when no active feeding is observed and the body is enclosed in a hardened cuticle. These physiological properties as well as undergoing processes resemble embryogenesis, since at the pupal stage organs and systems of the imago are formed. Therefore, recapitulation of the embryonic expression program during metamorphosis could be hypothesized. To assess this hypothesis at the transcriptome level, we have performed a comprehensive analysis of the developmental datasets available in the public domain. Indeed, for most datasets, the pupal gene expression resembles the embryonic rather than the larval pattern, interrupting gradual changes in the transcriptome. Moreover, changes in the transcriptome profile during the pupa-to-imago transition are positively correlated with those at the embryo-to-larvae transition, suggesting that similar expression programs are activated. Gene sets that change their expression level during the larval stage and revert it to the embryonic-like state during the metamorphosis are enriched with genes associated with metabolism and development. [ABSTRACT FROM AUTHOR]

Vaccinium bracteatum Thunb. (VBT) is widely distributed in the mountainous areas in eastern and southern China. VBT leaves have great medical value and can be used to stain rice to produce "Wumifan". Its fruits also contain rich nutrients. However, there has been limited attention to exploring the molecular content of VBT. Previously, we performed RNA-seq on three typical VBT fruits that were at various stages of ripening, although a reliable reference gene was lost in validation.In this study, we selected ten candidate reference genes based on previous studies and transcriptomics analyses. Subsequently, these genes were evaluated using a combination of methods, including geNorm, NormFinder, and Bestkeeper, with a comprehensive ranking assessment. As a result, we found that the actin2, NADH, and ADK genes have high reliability for analysing the expression levels of genes involved in fruit development. Furthermore, the transcript levels of 15 DEGs from transcriptomic analysis were assessed using NADH as a reference gene, and RT-qPCR data were highly consistent with the transcriptomic data. These results provide reliable reference genes for further studying gene expression, which will be beneficial for comprehensively exploring VBT. [ABSTRACT FROM AUTHOR]

The N-acylsulfonamide derivative, I942, represents the first non-cyclic nucleotide partial agonist of EPAC1. This was soon followed by the identification of the I942 analogues, PW0381, PW0521 and PWO577 and a series of benzofuran oxoacetic acid EPAC1 activators, SY006, SY007 and SY009. Protein interaction, cytotoxicity and EPAC1 activation assays applied here identify PWO577 and SY007 as being effective EPAC1 binders that are well tolerated in HUVECs at concentrations greater than 100 μM and up to 48 h incubation and are effective activators of transfected EPAC1 in U2OS cells. Using RNAseq in HUVECs we show that PWO577 and SY007 regulate approximately 11,000 shared genes, with only few differential gene changes being "off-target". The genes significantly regulated by both PWO577 and SY007 included a subset of genes normally associated with endothelial activation, including ICAM1, MMP1 and CCL2. Of these, only the expression of MMP1 was markedly increased at the protein level, as determined by LC–MS-based proteomics. Both PWO577 and SY007 suppressed IL-6-induced STAT3 activation and associated downstream gene expression, including inhibition of SOCS3, STAT3, IL6ST and JAK3 genes. Together these results demonstrate the utility of structurally distinct, specific and non-toxic EPAC1 activators. Future modifications will be aimed at eliminating the few noted off-target effects. [ABSTRACT FROM AUTHOR]

The cotton rat (Sigmodon) is the gold standard pre-clinical small animal model for respiratory viral pathogens, especially for respiratory syncytial virus (RSV). However, without a reference genome or a published transcriptome, studies requiring gene expression analysis in cotton rats are severely limited. The aims of this study were to generate a comprehensive transcriptome from multiple tissues of two species of cotton rats that are commonly used as animal models (Sigmodon fulviventer and Sigmodon hispidus), and to compare and contrast gene expression changes and immune responses to RSV infection between the two species. Transcriptomes were assembled from lung, spleen, kidney, heart, and intestines for each species with a contig N50 > 1600. Annotation of contigs generated nearly 120,000 gene annotations for each species. The transcriptomes of S. fulviventer and S. hispidus were then used to assess immune response to RSV infection. We identified 238 unique genes that are significantly differentially expressed, including several genes implicated in RSV infection (e.g., Mx2, I27L2, LY6E, Viperin, Keratin 6A, ISG15, CXCL10, CXCL11, IRF9) as well as novel genes that have not previously described in RSV research (LG3BP, SYWC, ABEC1, IIGP1, CREB1). This study presents two comprehensive transcriptome references as resources for future gene expression analysis studies in the cotton rat model, as well as provides gene sequences for mechanistic characterization of molecular pathways. Overall, our results provide generalizable insights into the effect of host genetics on host-virus interactions, as well as identify new host therapeutic targets for RSV treatment and prevention. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) stability substantially impacts steady-state gene expression levels in a cell. mRNA stability is strongly affected by codon composition in a translation-dependent manner across species, through a mechanism termed codon optimality. We have developed iCodon (www.iCodon.org), an algorithm for customizing mRNA expression through the introduction of synonymous codon substitutions into the coding sequence. iCodon is optimized for four vertebrate transcriptomes: mouse, human, frog, and fish. Users can predict the mRNA stability of any coding sequence based on its codon composition and subsequently generate more stable (optimized) or unstable (deoptimized) variants encoding for the same protein. Further, we show that codon optimality predictions correlate with both mRNA stability using a massive reporter library and expression levels using fluorescent reporters and analysis of endogenous gene expression in zebrafish embryos and/or human cells. Therefore, iCodon will benefit basic biological research, as well as a wide range of applications for biotechnology and biomedicine. [ABSTRACT FROM AUTHOR]