Single-cell analysis via manifold fitting: A framework for RNA clustering and beyond.

Single-cell RNA sequencing (scRNA-seq) data, susceptible to noise arising from biological variability and technical errors, can distort gene expression analysis and impact cell similarity assessments, particularly in heterogeneous populations. Current methods, including deep learning approaches, often struggle to accurately characterize cell relationships due to this inherent noise. To address these challenges, we introduce scAMF (Single-cell Analysis via Manifold Fitting), a framework designed to enhance clustering accuracy and data visualization in scRNA-seq studies. At the heart of scAMF lies the manifold fitting module, which effectively denoises scRNA-seq data by unfolding their distribution in the ambient space. This unfolding aligns the gene expression vector of each cell more closely with its underlying structure, bringing it spatially closer to other cells of the same cell type. To comprehensively assess the impact of scAMF, we compile a collection of 25 publicly available scRNA-seq datasets spanning various sequencing platforms, species, and organ types, forming an extensive RNA data bank. In our comparative studies, benchmarking scAMF against existing scRNA-seq analysis algorithms in this data bank, we consistently observe that scAMF outperforms in terms of clustering efficiency and data visualization clarity. Further experimental analysis reveals that this enhanced performance stems from scAMF's ability to improve the spatial distribution of the data and capture class-consistent neighborhoods. These findings underscore the promising application potential of manifold fitting as a tool in scRNA-seq analysis, signaling a significant enhancement in the precision and reliability of data interpretation in this critical field of study.

Genetics and metabolic responses of Artemisia annua L to the lake of phosphorus under the sparingly soluble phosphorus fertilizer: evidence from transcriptomics analysis.

The medicinal herb Artemisia annua L. is prized for its capacity to generate artemisinin, which is used to cure malaria. Potentially influencing the biomass and secondary metabolite synthesis of A. annua is plant nutrition, particularly phosphorus (P). However, most soil P exist as insoluble inorganic and organic phosphates, which results to low P availability limiting plant growth and development. Although plants have developed several adaptation strategies to low P levels, genetics and metabolic responses to P status remain largely unknown. In a controlled greenhouse experiment, the sparingly soluble P form, hydroxyapatite (Ca5OH(PO4)3/CaP) was used to simulate calcareous soils with low P availability. In contrast, the soluble P form KH2PO4/KP was used as a control. A. annua's morphological traits, growth, and artemisinin concentration were determined, and RNA sequencing was used to identify the differentially expressed genes (DEGs) under two different P forms. Total biomass, plant height, leaf number, and stem diameter, as well as leaf area, decreased by 64.83%, 27.49%, 30.47%, 38.70%, and 54.64% in CaP compared to KP; however, LC-MS tests showed an outstanding 37.97% rise in artemisinin content per unit biomass in CaP contrary to KP. Transcriptome analysis showed 2015 DEGs (1084 up-regulated and 931 down-regulated) between two P forms, including 39 transcription factor (TF) families. Further analysis showed that DEGs were mainly enriched in carbohydrate metabolism, secondary metabolites biosynthesis, enzyme catalytic activity, signal transduction, and so on, such as tricarboxylic acid (TCA) cycle, glycolysis, starch and sucrose metabolism, flavonoid biosynthesis, P metabolism, and plant hormone signal transduction. Meanwhile, several artemisinin biosynthesis genes were up-regulated, including DXS, GPPS, GGPS, MVD, and ALDH, potentially increasing artemisinin accumulation. Furthermore, 21 TF families, including WRKY, MYB, bHLH, and ERF, were up-regulated in reaction to CaP, confirming their importance in P absorption, internal P cycling, and artemisinin biosynthesis regulation. Our results will enable us to comprehend how low P availability impacts the parallel transcriptional control of plant development, growth, and artemisinin production in A. annua. This study could lay the groundwork for future research into the molecular mechanisms underlying A. annua's low P adaptation.

Reanalysis of single-cell data reveals macrophage subsets associated with the immunotherapy response and prognosis of patients with endometrial cancer.

Endometrial cancer (EC) is an aggressive gynecological malignancy with an increased incidence rate. The immune landscape crucially affects immunotherapy efficacy and prognosis in EC patients. Here, we characterized the distinct tumor microenvironment signatures of EC tumors by analyzing single-cell RNA sequencing data from Gene Expression Omnibus and bulk RNA sequencing data from The Cancer Genome Atlas, which were compared with normal endometrium. Three macrophage subsets were identified, and two of them showed tissue-specific distribution. One of the macrophage subsets was dominant in macrophages derived from EC and exhibited characteristic behaviors such as promoting tumor growth and metastasis. One of the other macrophage subsets was mainly found in normal endometrium and served functions related to antigen presentation. We also identified a macrophage subset that was found in both EC and normal endometrial tissue. However, the pathway and cellular cross-talk of this subset were completely different based on the respective origin, suggesting a tumor-related differentiation mechanism of macrophages. Additionally, the tumor-enriched macrophage subset was found to predict immunotherapy responses in EC. Notably, we selected six genes from macrophage subset markers that could predict the survival of EC patients, SCL8A1, TXN, ANXA5, CST3, CD74 and NANS, and constructed a prognostic signature. To verify the signature, we identified immunohistochemistry for the tumor samples of 83 EC patients based on the selected genes and further followed up with the survival of the patients. Our results provide strong evidence that the signature can effectively predict the prognosis of EC patients.

Analysis of the finasteride treatment and its withdrawal in the rat hypothalamus and hippocampus at whole-transcriptome level.

PURPOSE: As reported in patients treated for androgenetic alopecia with finasteride (i.e., a blocker of the enzyme 5 alpha-reductase) and in an animal model, side effects affecting sexual, psychiatric, neurological, and physical domains, may occur during the treatment and persist with drug suspension. The etiopathogenesis of these side effects has been poorly explored. Therefore, we performed a genome-wide analysis of finasteride effects in the brain of adult male rat. METHODS: Animals were treated (i.e., for 20 days) with finasteride (1mg/rat/day). 24 h after the last treatment and 1 month after drug suspension, RNA sequencing analysis was performed in hypothalamus and hippocampus. Data were analyzed by differential expression analysis and Gene-Set Enrichment Analyses (GSEA). RESULTS: Data obtained after finasteride treatment showed that 186 genes (i.e., 171 up- and 15 downregulated) and 19 (i.e., 17 up- and 2 downregulated) were differentially expressed in the hypothalamus and hippocampus, respectively. Differential expression analysis at the drug withdrawal failed to identify dysregulated genes. Several gene-sets were enriched in these brain areas at both time points. CONCLUSION: Some of the genes reported to be differentially expressed (i.e., TTR, DIO2, CLDN1, CLDN2, SLC4A5, KCNE2, CROT, HCRT, MARCKSL1, VGF, IRF2BPL) and GSEA, suggest a potential link with specific side effects previously observed in patients and in the animal model, such as depression, anxiety, disturbance in memory and attention, and sleep disturbance. These data may provide an important background for future experiments aimed at confirming the pathological role of these genes.

Unraveling intratumoral complexity in metastatic dermatofibrosarcoma protuberans through single-cell RNA sequencing analysis.

Dermatofibrosarcoma protuberans (DFSP) stands as a rare and locally aggressive soft tissue tumor, characterized by intricated molecular alterations. The imperative to unravel the complexities of intratumor heterogeneity underscores effective clinical management. Herein, we harnessed single-cell RNA sequencing (scRNA-seq) to conduct a comprehensive analysis encompassing samples from primary sites, satellite foci, and lymph node metastases. Rigorous preprocessing of raw scRNA-seq data ensued, and employing t-distributed stochastic neighbor embedding (tSNE) analysis, we unveiled seven major cell populations and fifteen distinct subpopulations. Malignant cell subpopulations were delineated using infercnv for copy number variation calculations. Functional and metabolic variations of diverse malignant cell populations across samples were deciphered utilizing GSVA and the scMetabolism R packages. Additionally, the exploration of differentiation trajectories within diverse fibroblast subpopulations was orchestrated through pseudotime trajectory analyses employing CytoTRACE and Monocle2, and further bolstered by GO analyses to elucidate the functional disparities across distinct differentiation states. In parallel, we segmented the cellular components of the immune microenvironment and verified the presence of SPP1+ macrophage, which constituted the major constituent in lymph node metastases. Remarkably, the CellChat facilitated a comprehensive intercellular communication analysis. This study culminates in an all-encompassing single-cell transcriptome atlas, propounding novel insights into the multifaceted nature of intratumor heterogeneity and fundamental molecular mechanisms propelling metastatic DFSP.

Senescence-associated secretory phenotypes in mesenchymal cells contribute to cytotoxic immune response in oral lichen planus.

Oral lichen planus is a chronic inflammatory condition that adversely affects the oral mucosa; however, its etiology remains elusive. Consequently, therapeutic interventions for oral lichen planus are limited to symptomatic management. This study provides evidence of the accumulation of senescent mesenchymal cells, CD8 + T cells, and natural killer cells in patients with oral lichen planus. We profiled the patients' tissues using the National Center for Biotechnology Information Gene Expression Omnibus database and found that senescence-related genes were upregulated in these tissues by gene set enrichment analysis. Immunohistochemical analysis showed increased senescent mesenchymal cells in the subepithelial layer of patients with oral lichen planus. Single-cell RNA-seq data retrieved from the Gene Expression Omnibus database of patients with oral lichen planus revealed that mesenchymal cells were marked by the upregulation of senescence-related genes. Cell-cell communication analysis using CellChat showed that senescent mesenchymal cells significantly influenced CD8 + T cells and natural killer cells via CXCL12-CXCR4 signaling, which is known to activate and recruit CD8 + T cells and NK cells. Finally, in vitro assays demonstrated that the secretion of senescence-associated factors from mesenchymal cells stimulated the activation of T cells and natural killer cells and promoted epithelial cell senescence and cytotoxicity. These findings suggest that the accumulation of mesenchymal cells with senescence-associated secretory phenotype may be a key driver of oral lichen planus pathogenesis.

The Transcription Factor ZmNAC89 Gene Is Involved in Salt Tolerance in Maize (Zea mays L.).

The NAC gene family has transcription factors specific to plants, which are involved in development and stress response and adaptation. In this study, ZmNAC89, an NAC gene in maize that plays a role in saline-alkaline tolerance, was isolated and characterized. ZmNAC89 was localized in the nucleus and had transcriptional activation activity during in vitro experiments. The expression of ZmNAC89 was strongly upregulated under saline-alkaline, drought and ABA treatments. Overexpression of the ZmNAC89 gene in transgenic Arabidopsis and maize enhanced salt tolerance at the seedling stage. Differentially expressed genes (DEGs) were then confirmed via RNA-sequencing analysis with the transgenic maize line. GO analyses showed that oxidation-reduction process-regulated genes were involved in ZmNAC89-mediated salt-alkaline stress. ZmNAC89 may regulate maize saline-alkali tolerance through the REDOX pathway and ABA signal transduction pathway. From 140 inbred maize lines, 20 haplotypes and 16 SNPs were found in the coding region of the ZmNAC89 gene, including the excellent haplotype HAP20. These results contribute to a better understanding of the response mechanism of maize to salt-alkali stress and marker-assisted selection during maize breeding.

Mapping developmental paths of monkey primordial germ-like cells differentiation from pluripotent stem cells by single cell ribonucleic acid sequencing analysis†.

The induction of primordial germ-like cells (PGCLCs) from pluripotent stem cells (PSCs) provides a powerful system to study the cellular and molecular mechanisms underlying germline specification, which are difficult to study in vivo. The studies reveal the existence of a species-specific mechanism underlying PGCLCs between humans and mice, highlighting the necessity to study regulatory networks in more species, especially in primates. Harnessing the power of single-cell RNA sequencing (scRNA-seq) analysis, the detailed trajectory of human PGCLCs specification in vitro has been achieved. However, the study of nonhuman primates is still needed. Here, we applied an embryoid body (EB) differentiation system to induce PGCLCs specification from cynomolgus monkey male and female PSCs, and then performed high throughput scRNA-seq analysis of approximately 40 000 PSCs and cells within EBs. We found that EBs provided a niche for PGCLCs differentiation by secreting growth factors critical for PGCLC specification, such as bone morphogenetic protein 2 (BMP2), BMP4, and Wnt Family Member 3. Moreover, the developmental trajectory of PGCLCs was reconstituted, and gene expression dynamics were revealed. Our study outlines the roadmap of PGCLC specification from PSCs and provides insights that will improve the differentiation efficiency of PGCLCs from PSCs.

Iron redistribution induces oxidative burst and resistance in maize against Curvularia lunata.

MAIN CONCLUSION: ΔClnps6 induced iron redistribution in maize B73 leaf cells and resulted in reactive oxygen species (ROS) burst to enhance plant resistance against Curvularia lunata. Iron is an indispensable co-factor of various crucial enzymes that are involved in cellular metabolic processes and energy metabolism in eukaryotes. For this reason, plants and pathogens compete for iron to maintain their iron homeostasis, respectively. In our previous study, ΔClnps6, the extracellular siderophore biosynthesis deletion mutant of Curvularia lunata, was sensitive to exogenous hydrogen peroxide and virulence reduction. However, the mechanism was not studied. Here, we report that maize B73 displayed highly resistance to ΔClnps6. The plants recruited more iron at cell wall appositions (CWAs) to cause ROS bursts. Intracellular iron deficiency induced by iron redistribution originated form up-regulated expression of genes involved in intracellular iron consumption in leaves and absorption in roots. The RNA-sequencing data also showed that the expression of respiratory burst oxidase homologue (ZmRBOH4) and NADP-dependent malic enzyme 4 (ZmNADP-ME4) involved in ROS production was up-regulated in maize B73 after ΔClnps6 infection. Simultaneously, jasmonic acid (JA) biosynthesis genes lipoxygenase (ZmLOX), allene oxide synthase (ZmAOS), GA degradation gene gibberellin 2-beta-dioxygenase (ZmGA2OX6) and ABA degradation genes abscisic acid hydroxylase (ZmABH1, ZmABH2) involved in iron homeostasis were up-regulated expression. Ferritin1 (ZmFER1) positive regulated maize resistance against C. lunata via ROS burst under Fe-limiting conditions. Overall, our results showed that iron played vital roles in activating maize resistance in B73-C. lunata interaction.

Single-cell RNA-sequencing atlas of bovine caudal intervertebral discs: Discovery of heterogeneous cell populations with distinct roles in homeostasis.

Back and neck pain are significant healthcare burdens that are commonly associated with pathologies of the intervertebral disc (IVD). The poor understanding of the cellular heterogeneity within the IVD makes it difficult to develop regenerative IVD therapies. To address this gap, we developed an atlas of bovine (Bos taurus) caudal IVDs using single-cell RNA-sequencing (scRNA-seq). Unsupervised clustering resolved 15 unique clusters, which we grouped into the following annotated partitions: nucleus pulposus (NP), outer annulus fibrosus (oAF), inner AF (iAF), notochord, muscle, endothelial, and immune cells. Analyzing the pooled gene expression profiles of the NP, oAF, and iAF partitions allowed us to identify novel markers for NP (CP, S100B, H2AC18, SNORC, CRELD2, PDIA4, DNAJC3, CHCHD7, and RCN2), oAF (IGFBP6, CTSK, LGALS1, and CCN3), and iAF (MGP, COMP, SPP1, GSN, SOD2, DCN, FN1, TIMP3, WDR73, and GAL) cells. Network analysis on subpopulations of NP and oAF cells determined that clusters NP1, NP2, NP4, and oAF1 displayed gene expression profiles consistent with cell survival, suggesting these clusters may uniquely support viability under the physiological stresses of the IVD. Clusters NP3, NP5, oAF2, and oAF3 expressed various extracellular matrix (ECM)-associated genes, suggesting their role in maintaining IVD structure. Lastly, transcriptional entropy and pseudotime analyses found that clusters NP3 and NP1 had the most stem-like gene expression signatures of the NP partition, implying these clusters may contain IVD progenitor cells. Overall, results highlight cell type diversity within the IVD, and these novel cell phenotypes may enhance our understanding of IVD development, homeostasis, degeneration, and regeneration.

Editome landscape of CCM-derived endothelial cells.

By regulating several phases of gene expression, RNA editing modifications contribute to maintaining physiological RNA expression levels. RNA editing dysregulation can affect RNA molecule half-life, coding/noncoding RNA interaction, alternative splicing, and circular RNA biogenesis. Impaired RNA editing has been observed in several pathological conditions, including cancer and Alzheimer's disease. No data has been published yet on the editome profile of endothelial cells (ECs) isolated from human cerebral cavernous malformation (CCM) lesions. Here, we describe a landscape of editome modifications in sporadic CCM-derived ECs (CCM-ECs) by comparing editing events with those observed in human brain microvascular endothelial cells (HBMECs). With a whole transcriptome-based variant calling pipeline, we identified differential edited genes in CCM-ECs that were enriched in pathways related to angiogenesis, apoptosis and cell survival, inflammation and, in particular, to thrombin signalling mediated by protease-activated receptors and non-canonical Wnt signalling. These pathways, not yet associated to CCM development, could be a novel field for further investigations on CCM molecular mechanisms. Moreover, enrichment analysis of differentially edited miRNAs suggested additional small noncoding transcripts to consider for development of targeted therapies.

Single-Cell RNA Sequencing Analysis for Oncogenic Mechanisms Underlying Oral Squamous Cell Carcinoma Carcinogenesis with Candida albicans Infection.

Oral squamous cell carcinoma (OSCC) carcinogenesis involves heterogeneous tumor cells, and the tumor microenvironment (TME) is highly complex with many different cell types. Cancer cell-TME interactions are crucial in OSCC progression. Candida albicans (C. albicans)-frequently pre-sent in the oral potentially malignant disorder (OPMD) lesions and OSCC tissues-promotes malignant transformation. The aim of the study is to verify the mechanisms underlying OSCC car-cinogenesis with C. albicans infection and identify the biomarker for the early detection of OSCC and as the treatment target. The single-cell RNA sequencing analysis (scRNA-seq) was performed to explore the cell subtypes in normal oral mucosa, OPMD, and OSCC tissues. The cell composi-tion changes and oncogenic mechanisms underlying OSCC carcinogenesis with C. albicans infec-tion were investigated. Gene Set Variation Analysis (GSVA) was used to survey the mechanisms underlying OSCC carcinogenesis with and without C. albicans infection. The results revealed spe-cific cell clusters contributing to OSCC carcinogenesis with and without C. albicans infection. The major mechanisms involved in OSCC carcinogenesis without C. albicans infection are the IL2/STAT5, TNFα/NFκB, and TGFß signaling pathways, whereas those involved in OSCC carcinogenesis with C. albicans infection are the KRAS signaling pathway and E2F target down-stream genes. Finally, stratifin (SFN) was validated to be a specific biomarker of OSCC with C. albicans infection. Thus, the detailed mechanism underlying OSCC carcinogenesis with C. albicans infection was determined and identified the treatment biomarker with potential precision medicine applications.

RNA sequencing analysis of Beauveria bassiana isolated from Ostrinia furnacalis identifies the pathogenic genes.

Beauveria bassiana (B. bassiana) is a broad-spectrum entomopathogenic species of fungi which is a natural enemy of Ostrinia furnacalis (O. furnacalis). Nevertheless, the precise mechanism of pathogenicity difference of B. bassiana strains on O. furnacalis has not been investigated before. In this study, two B. bassiana strains isolated from the infected O. furnacalis and exhibited different pathogenicity were chose to analyze the gene expression using RNA-sequencing analysis. To investigate the significantly differentially expressed genes (DEGs) of these two strains, total RNA was extracted and Cuffdiff software was applied to perform the significance analysis of the microarrays method. qRT-PCR was applied to verify the expression of DEGs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analyses were applied to evaluate the functions of DEGs. Data showed 72 up-regulated and 192 down-regulated genes in hyper-pathogenic strain ZK193 in comparison with hypo-pathogenic strain ZK203. Genes involved in fungal growth, sporulation and toxin production were up-regulated in hyper-pathogenic strain ZK193. GO enrichment analysis of DEGS showed that the most observably enriched biological processes of regulated genes were the single-organism process, the metabolic process, the cellular process and biological regulation. KEGG enrichment pathway demonstrated that the almost enriched groups were metabolic pathways, such as lipid metabolism, transport and catabolism, amino acid metabolism, and carbohydrate metabolism. In conclusion, these results will help us to further understand the reason why different B. bassiana strains exhibit different pathogenicity on the same host, even under the same conditions. In addition, transcriptome data will provide a theoretical basis for exploring latent virulence factors in the future.

Genome-wide RNA sequencing analysis reveals that IGF-2 attenuates memory decline, oxidative stress and amyloid plaques in an Alzheimer's disease mouse model (AD) by activating the PI3K/AKT/CREB signaling pathway.

OBJECTIVES: Alzheimer's Disease (AD), characterized by deficits in memory and cognition and by behavioral impairment, is a progressive neurodegenerative disorder that influences more than 47 million people worldwide. Currently, no available drug is able to stop AD progression. Therefore, novel therapeutic strategies need to be investigated. MEASUREMENTS: We analyzed the RNA sequencing data (RNA-seq) derived from the Gene Expression Omnibus (GEO) database to identify the differentially expressed mRNAs in AD. The AD mouse model Tg2576 was used to verify the effects of IGF-2. The Morris Water Maze was administered to test the role of IGF-2 in memory consolidation. In addition, we quantified cell apoptosis by the TUNEL assay. The levels of amyloid plaques and the levels of Aß40 and Aß42 in the hippocampus were also determined by immunohistochemistry and ELISA, respectively. RESULTS: RNA-seq analysis revealed that IGF-2 was remarkably reduced in AD. The expression of the upstream genes PI3K and AKT and the downstream gene CREB in the PI3K signaling pathway was significantly increased in the hippocampus of Tg2576 mice cells treated with IGF-2. The Morris water maze test showed that IGF-2 improved memory consolidation in Tg2576 mice. The activity of caspase-3 was decreased in Tg2576 mice treated with IGF-2. Amyloid plaques in the hippocampus were reduced, and the levels of Aß40 and Aß42 were decreased. The above effects of IGF-2 on AD were blocked when the PI3K signaling pathway inhibitor wortmannin was added. CONCLUSIONS: IGF-2 attenuates memory decline, oxidative stress, cell apoptosis and amyloid plaques in the AD mouse model Tg2576 by activating the PI3K/AKT/CREB signaling pathway.

Skeletal muscle in aged mice reveals extensive transformation of muscle gene expression.

BACKGROUND: Aging leads to decreased skeletal muscle function in mammals and is associated with a progressive loss of muscle mass, quality and strength. Age-related muscle loss (sarcopenia) is an important health problem associated with the aged population. RESULTS: We investigated the alteration of genome-wide transcription in mouse skeletal muscle tissue (rectus femoris muscle) during aging using a high-throughput sequencing technique. Analysis revealed significant transcriptional changes between skeletal muscles of mice at 3 (young group) and 24 (old group) months of age. Specifically, genes associated with energy metabolism, cell proliferation, muscle myosin isoforms, as well as immune functions were found to be altered. We observed several interesting gene expression changes in the elderly, many of which have not been reported before. CONCLUSIONS: Those data expand our understanding of the various compensatory mechanisms that can occur with age, and further will assist in the development of methods to prevent and attenuate adverse outcomes of aging.

Evaluation of logistic regression models and effect of covariates for case-control study in RNA-Seq analysis.

BACKGROUND: Next generation sequencing provides a count of RNA molecules in the form of short reads, yielding discrete, often highly non-normally distributed gene expression measurements. Although Negative Binomial (NB) regression has been generally accepted in the analysis of RNA sequencing (RNA-Seq) data, its appropriateness has not been exhaustively evaluated. We explore logistic regression as an alternative method for RNA-Seq studies designed to compare cases and controls, where disease status is modeled as a function of RNA-Seq reads using simulated and Huntington disease data. We evaluate the effect of adjusting for covariates that have an unknown relationship with gene expression. Finally, we incorporate the data adaptive method in order to compare false positive rates. RESULTS: When the sample size is small or the expression levels of a gene are highly dispersed, the NB regression shows inflated Type-I error rates but the Classical logistic and Bayes logistic (BL) regressions are conservative. Firth's logistic (FL) regression performs well or is slightly conservative. Large sample size and low dispersion generally make Type-I error rates of all methods close to nominal alpha levels of 0.05 and 0.01. However, Type-I error rates are controlled after applying the data adaptive method. The NB, BL, and FL regressions gain increased power with large sample size, large log2 fold-change, and low dispersion. The FL regression has comparable power to NB regression. CONCLUSIONS: We conclude that implementing the data adaptive method appropriately controls Type-I error rates in RNA-Seq analysis. Firth's logistic regression provides a concise statistical inference process and reduces spurious associations from inaccurately estimated dispersion parameters in the negative binomial framework.

RNA sequencing analysis reveals new findings of hyperbaric oxygen treatment on rats with acute carbon monoxide poisoning.

OBJECTIVE: To elucidate the altered gene network in the brains of carbon monoxide (CO) poisoned rats after treatment with hyperbaric oxygen (HBO2). METHODS: RNA sequencing (RNA-seq) analysis was performed to examine differentially expressed genes (DEGs) in brain tissue samples from nine male rats: a normal control group; a CO poisoning group; and an HBO2 treatment group (three rats/group). Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR were used for validation of the DEGs in another 18 male rats (six rats/group). RESULTS: RNA-seq revealed that two genes were upregulated (4.18 and 8.76 log to the base 2 fold change) (p⟨0.05) in the CO-poisoned rats relative to the control rats; two genes were upregulated (3.88 and 7.69 log to the base 2 fold change); and 23 genes were downregulated (3.49-15.12 log to the base 2 fold change) (p⟨0.05) in the brains of the HBO2-treated rats relative to the CO-poisoned rats. Target prediction of DEGs by gene network analysis and analysis of pathways affected suggested that regulation of gene expressions of dopamine metabolism and nitric oxide (NO) synthesis were significantly affected by CO poisoning and HBO2 treatment. Results of RT-PCR and real-time quantitative PCR indicated that four genes (Pomc, GH-1, Pr1 and Fshß) associated with hormone secretion in the hypothalamic-pituitary system have potential as markers for prognosis of CO. CONCLUSION: This study is the first RNA-seq analysis profile of HBO2 treatment on rats with acute CO poisoning. It concludes that changes of hormone secretion in the hypothalamic-pituitary system, dopamine metabolism and NO synthesis involved in brain damage and behavior abnormalities after CO poisoning and HBO2 therapy may regulate these changes.

Formononetin attenuates psoriasiform inflammation by regulating interferon signaling pathway.

BACKGROUND: Psoriasis is a long-lasting, inflammatory, continuous illness caused through T cells and characterized mainly by abnormal growth and division of keratinocytes. Currently, corticosteroids are the preferred option. However, prolonged use of traditional topical medication can lead to adverse reactions and relapse, presenting a significant therapeutic obstacle. Improved alternative treatment options are urgently required. Formononetin (FMN) is a representative component of isoflavones in Huangqi (HQ) [Astragalus membranaceus (Fisch.) Bge.]. It possesses properties that reduce inflammation, combat oxidation, inhibit tumor growth, and mimic estrogen. Although FMN has been shown to ameliorate skin barrier devastation via regulating keratinocyte apoptosis and proliferation, there are no reports of its effectiveness in treating psoriasis. OBJECTIVE: Through transcriptomics clues and experimental investigation, we aimed to elucidate the fundamental mechanisms underlying FMN's action on psoriasis. MATERIALS AND METHODS: Cell viability was examined using CCK8 assay in this study. The results of analysis of differentially expressed genes (DEGs) between FMN-treated HaCaT cells and normal HaCaT cells using RNA-sequencing (RNA-seq) were presented on volcano plots and heatmap. Enrichment analysis was conducted on DEGs using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), and results were validated through RT-qPCR verification. After 12 days of FMN treatment in psoriasis mouse model, we gauged the PASI score and epidermis thickness. A variety of techniques were used to assess FMN's effectiveness on inhibiting inflammation and proliferation related to psoriasis, including RT-qPCR, HE staining, western blot, and immunohistochemistry (IHC). RESULTS: The findings indicated that FMN could suppress the growth of HaCaT cells using CCK8 assay (with IC50 = 40.64 uM) and 20 uM FMN could reduce the level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to the greatest extent. FMN-treated HaCaT cells exhibited 985 up-regulated and 855 down-regulated DEGs compared to normal HaCaT cells. GO analysis revealed that DEGs were linked to interferon (IFN) signaling pathway. Furthermore, FMN improved pathological features, which encompassed decreased erythema, scale, and thickness scores of skin lesions in psoriasis mouse model. In vivo experiments confirmed that FMN down-regulated expression of IFN-α, IFN-ß, IFN-γ, decreased secretion of TNF-α and IL-17 inflammatory factors, inhibited expression of IFN-related chemokines included Cxcl9, Cxcl10, Cxcl11 and Cxcr3 and reduced expression of transcription factors p-STAT1, p-STAT3 and IFN regulatory factor 1 (IRF1) in the imiquimod (IMQ) group. CONCLUSIONS: In summary, these results suggested that FMN played an anti-inflammatory and anti-proliferative role in alleviating psoriasis by inhibiting IFN signaling pathway, and FMN could be used as a potential therapeutic agent.

Cryptococcus neoformans Transcriptome Analysis to Identify Differentially Expressed Genes Using the STAR Pipeline in CyVerse Discovery Environment.

RNA sequencing is a next-generation sequencing approach that may be used to investigate many aspects of gene expression changes between cells. Analysis of the data is typically a multistep process using several bioinformatics tools. The following protocol utilizes a reliable pipeline for identifying differentially expressed genes among samples of Cryptococcus neoformans that is approachable for the adventurous beginner.

Single-cell RNA sequencing analysis provides novel insights into the role of apoptosis-related genes in muscle aging.

OBJECTIVE: This study employed a comprehensive single-cell analysis approach to explore the role of cell apoptosis-related genes in muscle aging. METHODS: The single-cell RNA sequencing data from the GSE143704 dataset were used to identify distinct cell clusters and assess gene expression patterns related to apoptosis activation. The "limma" package was used to identify hub genes, after which we performed Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to identify relevant pathways. Additionally, Gene Set Enrichment Analysis(GSEA) and Gene Set Variation Analysis (GSVA) were used to uncover relevant biological pathways. The Receiver Operating Characteristic Curve (ROC) was used to evaluate the diagnostic value of the hub genes. Single-sample Gene Set Enrichment Analysis (ssGSEA) was used to analyze the immune cell infiltration levels. RESULTS: Single-cell sequencing data from muscle aging patients allowed the identification of various cell types, including epithelial cells, adipocytes, and tissue-resident macrophages. By conducting a differential expression analysis that intersected active and nonactive apoptosis, as well as comparing elderly and young samples, a total of 22 hub genes were identified (p < 0.05). The 22 hub genes have discriminative ability as potential biomarkers for diagnosing muscle aging. The enrichment analysis indicated that these genes were closely associated with diverse pathways, including "response to UV-B" and "extracellular matrix organization" (p < 0.05). Furthermore, GSEA and GSVA indicated that multiple pathways emerged-for example, the "complement and coagulation cascades", "proteasome", "insulin signaling pathway", and "MAPK signaling pathway". Additionally, the analysis of immune cell infiltration revealed positive correlations between most of the hub genes and immune cells. CONCLUSION: Our study identified 22 apoptosis-related genes involved in muscle aging and indicated their potential diagnostic value. These findings offer a novel perspective on the pathogenesis of muscle aging and present potential targets for therapeutic interventions.