Integrated Transcriptome Analysis Research Articles

Integrated analysis of multiple metabolome and transcriptome revealed the accumulation of flavonoids and associated molecular regulation mechanisms in Rubus chingii Hu at different developmental stages

The traditional Chinese herb Rubus chingii Hu (R. chingii) is widely used in clinical practice due to its beneficial effects. Flavonoids are the important class of pharmacological substances in R. chingii, however, the molecular mechanism underlying the differences in active flavonoid contents in R. chingii at different developmental stages remain poorly understood. In this experiment, we selected four developmental stages (GG, GY, YR, RR) of R. chingii as the research material. We studied the untargeted and targeted metabolic profiles of flavonoids in different periods of R. chingii, combining full-length and comparative transcriptome analyses. Functional analyses were conducted on genes implicated in flavonoid differences. GG and RR displayed relatively higher and lower contents of flavonols, flavones, flavanols, flavanones, and isoflavonoid, respectively. RNA-seq analyses showed structural genes such as RcPAL, RcC4H, Rc4CL, RcCHS, RcCHI, RcF3H, RcF3′H, and RcFLS in flavonoid biosynthesis pathway were upregulated in GG, which were essential for the accumulation flavanones, flavones, and flavonols (effective components). qRT-PCR analyses investigated that six structural genes RcCHI, RcF3H, 2 RcCHS, and 2 Rc4CL, two TFs RcMYB308 and RcMYB123 had a consistent expression pattern with which in transcriptome. Also, an interaction network showed that the RcMYB308 could positively regulate Ka3R, Qu, Qu3G, AS, Hy, Ti through RcF3H. Furthermore, Subcellular localization analysis revealed that RcMYB308 was localization to the nucleus. In tobacco, RcMYB308 was overexpressed, resulting in higher flavonoids, RcF3H, RcF3′H, RcCHI, and RcFLS. RcMYB308 upregulated RcF3H in dual-luciferase assays. These results provide new insights for further understanding the molecular mechanism regulating flavonol biosynthesis in R. chingii fruit, and also provide a potential MYB regulator for molecular breeding of R. chingii.

CAG repeat expansion in the Huntington's disease gene shapes linear and circular RNAs biogenesis.

Alternative splicing (AS) appears to be altered in Huntington's disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, a significant proportion (36%) of the aberrantly spliced isoforms are not-functional and meant to non-sense mediated decay (NMD). The expanded Htt CAG repeats further reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Integrative transcriptomic analyses unveil a network of transcriptionally altered micro-RNAs and RNA-binding proteins (Celf, hnRNPs, Ptbp, Srsf, Upf1, Ythd2) which might influence the AS machinery, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might alter neural fitness, contributing to HD pathogenesis.

Integrative Transcriptome and Proteome Analyses Elucidate the Mechanism of Lens-Induced Myopia in Mice.

The purpose of this study was to investigate the underlying molecular mechanism of lens-induced myopia (LIM) through transcriptome and proteome analyses with a modified mouse myopia model. Four-week-old C57BL/6J mice were treated with a homemade newly designed -25 diopter (D) lens mounting by a 3D printing pen before right eyes for 4 weeks. Refraction (RE) and axial dimensions were measured every 2 weeks. Retinas were analyzed by RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, and STRING databases were used to identify significantly affected pathways in transcriptomic and proteomic data sets. Western blot was used to detect the expression of specific proteins. The modified model was accessible and efficient. Mice displayed a significant myopic shift (approximately 8 D) following 4 weeks' of lens treatment. Through transcriptomics and proteomics analysis, we elucidated 175 differently expressed genes (DEGs) and 646 differentially expressed proteins (DEPs) between binoculus. The transcriptomic and proteomic data showed a low correlation. Going over the mRNA protein matches, insulin like growth factor 2 mRNA binding protein 1 (Igf2bp1) was found to be a convincing biomarker of LIM, which was confirmed by Western blot. RNA-seq and proteome profiling confirmed that these two "omics" data sets complemented one another in KEGG pathways annovation. Among these, metabolic and human diseases pathways were considered to be correlated with the LIM forming process. The newly constructed LIM model provides a useful tool for future myopia research. Combining transcriptomic and proteomic analysis may potentially brighten the prospects of novel therapeutic targets for patients with myopia.

3β-Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1.

3β-hydroxy-12-oleanen-27-oic acid (ATA), a cytotoxic oleanane triterpenoid with C14-COOH isolated from the rhizome of Astilbe chinensis, has been previously proven to possess antitumor activity and may be a promising antitumor agent. However, its molecular mechanisms of antitumor action were still unclear. This study explored the underlying mechanisms of cytotoxicity and potential target of ATA against human colorectal cancer HCT116 cells via integrative analysis of transcriptomics and network pharmacology in combination with in vitro and in vivo experimental validations. ATA significantly inhibited the proliferation of HCT116 cells in a concentration- and time-dependent manner and induced the cell cycle arrest at the G0/G1 phase, apoptosis, autophagy, and ferroptosis. Transcriptomic analysis manifested that ATA regulated mRNA expression of the genes related to cell proliferation, cell cycle, and cell death in HCT116 cells. The integrated analysis of transcriptomics, network pharmacology, and molecular docking revealed that ATA exerted cytotoxic activity via interactions with FDFT1, PPARA, and PPARG. Furthermore, FDFT1 was verified to be an upstream key target mediating the antiproliferative effect of ATA against HCT116 cells. Of note, ATA remarkably suppressed the growth of HCT116 xenografts in nude mice and displayed an apparent attenuation of FDFT1 in tumor tissues accompanied by the alteration of the biomarkers of autophagy, cell cycle, apoptosis, and ferroptosis. These results demonstrate that ATA exerted in vitro and in vivo antiproliferative effects against HCT116 cells through inducing cell apoptosis, autophagy, and ferroptosis via targeting FDFT1.

Lanhuashen stimulates the positive cross-regulation mediated by the S1P axis to ameliorate the disorder of glucolipid metabolism induced by the high sucrose diet in Drosophila melanogaster

Ethnopharmacological relevanceHerba Wanlenbergiae, named ‘Lanhuashen’ (LHS) in Chinese, is derived from the dried herba of Wahlenbergia marginata (Thunb.) A.DC. It is an abundant resource that has been used in traditional Chinese medicine (TCM) for over 600 years. LHS has the effects of enriching consumptive disease and relieving deficient heat, consistent with the therapy for type 2 diabetes mellitus (T2DM) in TCM. As the basic remedy of Yulan Jiangtang capsules, a listed Chinese medicine specifically for treating T2DM, LHS is a potential candidate for an anti-T2DM drug. However, due to the lack of pharmacodynamic studies and chemical component analysis, the application and development of LHS as a treatment for T2DM have been hindered. Aim of the studyTo evaluate the regulation of the disorder of glucolipid metabolism using LHS extracts and its therapeutic potential in T2DM. Materials and methodsChemical components in LHS extracts were analysed using UPLC-Q Exactive-Orbitrap-MS. Subsequently, high sucrose diet (HSD)-induced Drosophila melanogaster were used as suitable models for T2DM in vivo. Behavioural and biochemical tests were performed to evaluate the regulation of the disorder of glucolipid metabolism using LHS in T2DM flies. Furthermore, integrative metabolomic and transcriptomic analysis was applied to reveal the specific effects of LHS extracts on metabolites and genes. Meanwhile, bioinformatic analysis was carried out to predict the targeted transcription factors (TFs) and potentially effective components of LHS extracts. ResultsWe redefined the chemical profile of LHS with 76 identified chemical components, including 65 chemical components for the first time. As indicated by decreased trehalose, glucose and triglyceride levels and increased total protein levels, LHS extracts were perceived to alleviate the disorder of glucolipid metabolism in HSD-induced T2DM fruit flies. Integrative metabolomic and transcriptomic analysis revealed that LHS extracts eliminated the accumulation of sphingolipids and subsequently stimulated the positive cross-regulation mediated by the sphingosine 1-phosphate (S1P) axis, resulting in the activation of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) signalling pathway and inhibition of lysosome-mediated apoptosis. Bioinformatic analysis revealed that the upstream TFs, transcriptional enhancer factor TEF-5 (TEAD3) and peroxisome proliferator-activated receptor alpha (PPARA), were the potential targets of atractylenolide III, dihydrokaempferol and syringaldehyde, the potentially effective components of LHS extracts. Therefore, this TF network was plausibly the basis for the efficacy. ConclusionsLHS extracts broadly modulated TF-dependent gene expression and subsequently stimulated the positive cross-regulation mediated by the S1P axis to ameliorate the disorder of glucolipid metabolism. Our study provides critical evidence considering LHS as a potential drug candidate for T2DM, inspiring the discovery and development of innovative therapeutic agents based on the cross-regulation mediated by the S1P axis for treating T2DM and related complications.

Identication and validation of cell senescence biomarkers in idiopathic pulmonary hypertension via integrated transcriptome analyses and machine learning

BackgroundIdiopathic pulmonary hypertension (IPAH) is a rare and severe disease that affects the pulmonary vasculature. As the diagnosis of IPAH requires invasive right heart catheterization surgery, early detection of this condition is notoriously challenging. Therefore, it is of utmost importance to investigate biomarkers present in peripheral blood that could aid physicians in the early identification and management of IPAH. MethodWe speculate that cellular senescence may be involved in the occurrence and development of IPAH through various pathways. In this study, we utilized integrated transcriptome analyses and machine learning-based approach to develop a diagnostic model for IPAH cell senescence. To select genetic features, we employed two machine learning algorithms: the Least Absolute Shrinkage and Selection Operator (LASSO) and Random Forest (RF). Additionally, we validated our findings through both external data sets and qRT-PCR experiments. ResultsThe resulting diagnostic nomogram was able to identify five important biomarkers that can aid in the diagnosis of IPAH, including TNFRSF1B, CCL16, GCLM, IL15, and SOD1. These genes are primarily associated with the immune system, as well as with cell senescence and apoptosis. ConclusionOur study demonstrates the utility of machine learning algorithms in making accurate diagnoses of IPAH, providing clinicians with a more directed approach to the diagnosis and treatment of this disease.

The impact of Parkinson's disease-associated gut microbiota on the transcriptome in Drosophila.

Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people, and many studies have confirmed that the disorder of gut microbiota is involved in the pathophysiological process of PD. However, the molecular mechanism of gut microbiota in regulating the pathogenesis of PD is still lacking. In this study, to investigate the impact of PD-associated gut microbiota on host transcriptome, we established various PD models with fecal microbiota transplantation (FMT) in the model organism Drosophila followed by integrative data analysis of microbiome and transcriptome. We first constructed rotenone-induced PD models in Drosophila followed by FMT in different groups. Microbial analysis by 16S rDNA sequencing showed that gut microbiota from PD Drosophila could affect bacterial structure of normal Drosophila, and gut microbiota from normal Drosophila could affect bacterial structure of PD Drosophila. Transcriptome analysis revealed that PD-associated gut microbiota influenced expression patterns of genes enriched in neuroactive ligand-receptor interaction, lysosome, and diverse metabolic pathways. Importantly, to verify our findings, we transplanted Drosophila with fecal samples from clinical PD patients. Compared to the control, Drosophila transplanted with fecal samples from PD patients had reduced microbiota Acetobacter and Lactobacillus, and differentially expressed genes enriched in diverse metabolic pathways. In summary, our results reveal the influence of PD-associated gut microbiota on host gene expression, and this study can help better understand the link between gut microbiota and PD pathogenesis through gut-brain axis. IMPORTANCE Gut microbiota plays important roles in regulating host gene expression and physiology through complex mechanisms. Recently, it has been suggested that disorder of gut microbiota is involved in the pathophysiological process of Parkinson's disease (PD). However, the molecular mechanism of gut microbiota in regulating the pathogenesis of PD is still lacking. In this study, to investigate the impact of PD-associated gut microbiota on host transcriptome, we established various PD models with fecal microbiota transplantation in the model organism Drosophila followed by integrative data analysis of microbiome and transcriptome. We also verified our findings by transplanting Drosophila with fecal samples from clinical PD patients. Our results demonstrated that PD-associated gut microbiota can induce differentially expressed genes enriched in diverse metabolic pathways. This study can help better understand the link between gut microbiota and PD pathogenesis through gut-brain axis.

Integrative transcriptomic analysis deciphering the role of rice bHLH transcription factor Os04g0301500 in mediating responses to biotic and abiotic stresses.

Understanding the signaling pathways activated in response to these combined stresses and their crosstalk is crucial to breeding crop varieties with dual or multiple tolerances. However, most studies to date have predominantly focused on individual stress factors, leaving a significant gap in understanding plant responses to combined biotic and abiotic stresses. The bHLH family plays a multifaceted regulatory role in plant response to both abiotic and biotic stresses. In order to comprehensively identify and analyze the bHLH gene family in rice, we identified putative OsbHLHs by multi-step homolog search, and phylogenic analysis, molecular weights, isoelectric points, conserved domain screening were processed using MEGAX version 10.2.6. Following, integrative transcriptome analysis using 6 RNA-seq data including Xoo infection, heat, and cold stress was processed. The results showed that 106 OsbHLHs were identified and clustered into 17 clades. Os04g0301500 and Os04g0489600 are potential negative regulators of Xoo resistance in rice. In addition, Os04g0301500 was involved in non-freezing temperatures (around 4°C) but not to 10°C cold stresses, suggesting a complex interplay with temperature signaling pathways. The study concludes that Os04g0301500 may play a crucial role in integrating biotic and abiotic stress responses in rice, potentially serving as a key regulator of plant resilience under changing environmental conditions, which could be important for further multiple stresses enhancement and molecular breeding through genetic engineering in rice.

Elevated expression of glycolytic genes as a prominent feature of early-onset preeclampsia: insights from integrative transcriptomic analysis.

Introduction: Preeclampsia (PE), a notable pregnancy-related disorder, leads to 40,000+ maternal deaths yearly. Recent research shows PE divides into early-onset (EOPE) and late-onset (LOPE) subtypes, each with distinct clinical features and outcomes. However, the molecular characteristics of various subtypes are currently subject to debate and are not consistent. Methods: We integrated transcriptomic expression data from a total of 372 placental samples across 8 publicly available databases via combat algorithm. Then, a variety of strategies including Random Forest Recursive Feature Elimination (RF-RFE), differential analysis, oposSOM, and Weighted Correlation Network Analysis were employed to identify the characteristic genes of the EOPE and LOPE subtypes. Finally, we conducted in vitro experiments on the key gene HK2 in HTR8/SVneo cells to explore its function. Results: Our results revealed a complex classification of PE placental samples, wherein EOPE manifests as a highly homogeneous sample group characterized by hypoxia and HIF1A activation. Among the core features is the upregulation of glycolysis-related genes, particularly HK2, in the placenta-an observation corroborated by independent validation data and single-cell data. Building on the pronounced correlation between HK2 and EOPE, we conducted in vitro experiments to assess the potential functional impact of HK2 on trophoblast cells. Additionally, the LOPE samples exhibit strong heterogeneity and lack distinct features, suggesting a complex molecular makeup for this subtype. Unsupervised clustering analysis indicates that LOPE likely comprises at least two distinct subtypes, linked to cell-environment interaction and cytokine and protein modification functionalities. Discussion: In summary, these findings elucidate potential mechanistic differences between the two PE subtypes, lend support to the hypothesis of classifying PE based on gestational weeks, and emphasize the potential significant role of glycolysis-related genes, especially HK2 in EOPE.

Dietary supplementation of Patchouli oil increases the resistance against Streptococcus agalactiae infection in GIFT tilapia as revealed by transcriptome and 16s amplicon profiling

Large-scale disease outbreaks that caused by Streptococcus agalactiae (SA) infection have dramatically affected tilapia production worldwide. Patchouli oil (PO) is a traditional Chinese herbal medicine in treating gastrointestinal symptoms and is considered to be a safe and efficient candidate to treat SA infection in tilapia. In this study, we first investigated the changes of intestinal microbiota and host responses in GIFT tilapia after a 28-day dietary supplementation of different concentrations of PO (basic diet, 5 μL PO/g diet, or 15 μL PO/g diet) and followed by SA infection using an integrative analysis of transcriptome and 16 S amplicon profiling. We observed that dietary supplementation of 5 μL PO/g diet improved food intake and growth in tilapia. Histopathological observation confirmed there were increased surface area of intestinal villi and generally decreased ROS as revealed by the MAD, GSH-PX, and T-SOD activities in the tilapia intestines in response to dietary PO supplementation. The intestinal microbial diversities and community structures were significantly affected, and the colonization of probiotics (e.g., Cetobacterium and clostridium_ sensu_stricto_1) in the mucosa was improved in the dietary PO groups. The relative abundance of Streptococcus (0.033%) in the 15 μL PO/g dietary group was much lower than that in the control group (basic diet; 0.86%). A large number of DEGs were enriched in the immune-related pathways, e.g., lysosomal lumen, MHC protein complex, and toll-like receptor 2 signaling pathway. Our findings suggested that dietary supplementation of PO enhanced intestinal physical barriers, increased the colonization of probiotic bacteria and improved intestinal immunity in tilapia. This provides novel knowledge on PO as a dietary supplement to improve intestinal health against SA invasion in tilapia.

Integrated Transcriptome Analysis of Long Noncoding RNA and mRNA in Developing and Aging Mouse Retina

Mice have emerged as a widely employed model for investigating various retinal diseases. However, the availability of comprehensive datasets capturing the entire developmental and aging stages of the mouse retina, particularly during the elderly period, encompassing integrated lncRNA and mRNA expression profiles, is limited. In this study, we assembled a total of 18 retina samples from mice across 6 distinct stages of development and aging (5 days, 3 weeks, 6 weeks, 10 weeks, 6 months, and 15 months) to conduct integrated lncRNA and mRNA sequencing analysis. This invaluable dataset offers a comprehensive transcriptomic resource of mRNA and lncRNA expression profiles during the natural progression of retinal development and aging. The discoveries stemming from this investigation will significantly contribute to the elucidation of the underlying molecular mechanisms associated with various retinal diseases, such as congenital retinal dysplasia and retinal degenerative diseases.

Integrative Analysis of Metabolome and Transcriptome Revealed Lutein Metabolism Contributed to Yellow Flower Formation in Prunus mume.

Prunus mume is a famous ornamental woody tree with colorful flowers. P. mume with yellow flowers is one of the most precious varieties. Regretfully, metabolites and regulatory mechanisms of yellow flowers in P. mume are still unclear. This hinders innovation of flower color breeding in P. mume. To elucidate the metabolic components and molecular mechanisms of yellow flowers, we analyzed transcriptome and metabolome between 'HJH' with yellow flowers and 'ZLE' with white flowers. Comparing the metabolome of the two varieties, we determined that carotenoids made contributions to the yellow flowers rather than flavonoids. Lutein was the key differential metabolite to cause yellow coloration of 'HJH'. Transcriptome analysis revealed significant differences in the expression of carotenoid cleavage dioxygenase (CCD) between the two varieties. Specifically, the expression level of PmCCD4 was higher in 'ZLE' than that in 'HJH'. Moreover, we identified six major transcription factors that probably regulated PmCCD4 to affect lutein accumulation. We speculated that carotenoid cleavage genes might be closely related to the yellow flower phenotype in P. mume. Further, the coding sequence of PmCCD4 has been cloned from the 'HJH' petals, and bioinformatics analysis revealed that PmCCD4 possessed conserved histidine residues, ensuring its enzymatic activity. PmCCD4 was closely related to PpCCD4, with a homology of 98.16%. Instantaneous transformation analysis in petal protoplasts of P. mume revealed PmCCD4 localization in the plastid. The overexpression of PmCCD4 significantly reduced the carotenoid content in tobacco plants, especially the lutein content, indicating that lutein might be the primary substrate for PmCCD4. We speculated that PmCCD4 might be involved in the cleavage of lutein in plastids, thereby affecting the formation of yellow flowers in P. mume. This work could establish a material and molecular basis of molecular breeding in P. mume for improving the flower color.

Integrative analysis of genome and transcriptome reveal the genetic basis of high temperature tolerance in pleurotus giganteus (Berk. Karun & Hyde)

BackgroundPleurotus giganteus is a commonly cultivated mushroom with notable high temperature resistance, making it significant for the growth of the edible fungi industry in the tropics. Despite its practical importance,, the genetic mechanisms underlying its ability to withstand high temperature tolerance remain elusive.ResultsIn this study, we performed high-quality genome sequencing of a monokaryon isolated from a thermotolerant strain of P. giganteus. The genome size was found to be 40.11 Mb, comprising 17 contigs and 13,054 protein-coding genes. Notably, some genes related to abiotic stress were identified in genome, such as genes regulating heat shock protein, protein kinase activity and signal transduction. These findings provide valuable insights into the genetic basis of P. giganteus’ high temperature resistance. Furthermore, the phylogenetic tree showed that P. giganteus was more closely related to P. citrinopileatus than other Pleurotus species. The divergence time between Pleurotus and Lentinus was estimated as 153.9 Mya, and they have a divergence time with Panus at 168.3 Mya, which proved the taxonomic status of P. giganteus at the genome level. Additionally, a comparative transcriptome analysis was conducted between mycelia treated with 40 °C heat shock for 18 h (HS) and an untreated control group (CK). Among the 2,614 differentially expressed genes (DEGs), 1,303 genes were up-regulated and 1,311 were down-regulated in the HS group. The enrichment analysis showed that several genes related to abiotic stress, including heat shock protein, DnaJ protein homologue, ubiquitin protease, transcription factors, DNA mismatch repair proteins, and zinc finger proteins, were significantly up-regulated in the HS group. These genes may play important roles in the high temperature adaptation of P. giganteus. Six DEGs were selected according to fourfold expression changes and were validated by qRT-PCR, laying a good foundation for further gene function analysis.ConclusionOur study successfully reported a high-quality genome of P. giganteus and identified genes associated with high-temperature tolerance through an integrative analysis of the genome and transcriptome. This study lays a crucial foundation for understanding the high-temperature tolerance mechanism of P. giganteus, providing valuable insights for genetic modification of P. giganteus strains and the development of high-temperature strains for the edible fungus industry, particularly in tropical regions.

Integrated Analysis of the Transcriptome and Metabolome of Brassica rapa Revealed Regulatory Mechanism under Heat Stress.

Affected by global warming; heat stress is the main limiting factor for crop growth and development. Brassica rapa prefers cool weather, and heat stress has a significant negative impact on its growth, development, and metabolism. Understanding the regulatory patterns of heat-resistant and heat-sensitive varieties under heat stress can help deepen understanding of plant heat tolerance mechanisms. In this study, an integrative analysis of transcriptome and metabolome was performed on the heat-tolerant ('WYM') and heat-sensitive ('AJH') lines of Brassica rapa to reveal the regulatory networks correlated to heat tolerance and to identify key regulatory genes. Heat stress was applied to two Brassica rapa cultivars, and the leaves were analyzed at the transcriptional and metabolic levels. The results suggest that the heat shock protein (HSP) family, plant hormone transduction, chlorophyll degradation, photosynthetic pathway, and reactive oxygen species (ROS) metabolism play an outstanding role in the adaptation mechanism of plant heat tolerance. Our discovery lays the foundation for future breeding of horticultural crops for heat resistance.

Integrative transcriptomic and metabolomic analysis in mice reveals the mechanism by which ginseng stem-leaf saponins enhance mucosal immunity induced by a porcine epidemic diarrhea virus vaccination

Porcine epidemic diarrhea virus (PEDV) is a main cause of severe enteric disease in piglets, leading to millions of dollars lost annually in the global pig industry. Parenteral vaccination is limited in generating sufficient mucosal immunity, which is crucial for early defense against PEDV. Here, we orally administered ginseng stem-leaf saponins (GSLS) to mice before parenteral vaccination and found that GSLS significantly enhanced the phagocytosis of dendritic cells, promoted the activities of CD4+ T cells and increased PEDV-specific IgA antibodies in the intestinal mucosa. Transcriptomic results showed that the altered genes following GSLS treatment were mostly related to the immune response and metabolism. In addition, integrated analysis of the transcriptome and metabolome revealed that the mechanism by which GSLS enhances mucosal immunity may be associated with progesterone-related pathways. Further studies are needed to explore the detailed molecular mechanisms.

Integrative single-cell transcriptome analysis reveals immune suppressive landscape in the anaplastic thyroid cancer.

The tumor immune microenvironment (TIME) in ATC is a complex and diverse ecosystem. It is essential to have a comprehensive understanding to improve cancer treatment and prognosis. However, TIME of ATC and the dynamic changes with PTC has not been revealed at the single-cell level. Here, we performed an integrative single-cell analysis of PTC and ATC primary tumor samples. We found that immunosuppressive cells and molecules dominated the TIME in ATC. Specifically, the level of infiltration of exhausted CD8+ T cells, and M2 macrophages was increased, and that of NK cells, B cells, and M1 macrophages was decreased. The cytotoxicity of CD8+ T cells, γδT cells, and NK cells was decreased, and immune checkpoint molecules, such as LAG3, PD1, HAVCR2, and TIGIT were highly expressed in ATC. Our findings contribute to the comprehension of TIME in both PTC and ATC, offering insights into the immunosuppressive factors specifically associated with ATC. Targeting these immunosuppressive factors may activate the anti-tumor immune response in ATC.

Single cell multiomic analysis reveals diabetes-associated β-cell heterogeneity driven by HNF1A

Broad heterogeneity in pancreatic β-cell function and morphology has been widely reported. However, determining which components of this cellular heterogeneity serve a diabetes-relevant function remains challenging. Here, we integrate single-cell transcriptome, single-nuclei chromatin accessibility, and cell-type specific 3D genome profiles from human islets and identify Type II Diabetes (T2D)-associated β-cell heterogeneity at both transcriptomic and epigenomic levels. We develop a computational method to explicitly dissect the intra-donor and inter-donor heterogeneity between single β-cells, which reflect distinct mechanisms of T2D pathogenesis. Integrative transcriptomic and epigenomic analysis identifies HNF1A as a principal driver of intra-donor heterogeneity between β-cells from the same donors; HNF1A expression is also reduced in β-cells from T2D donors. Interestingly, HNF1A activity in single β-cells is significantly associated with lower Na+ currents and we nominate a HNF1A target, FXYD2, as the primary mitigator. Our study demonstrates the value of investigating disease-associated single-cell heterogeneity and provides new insights into the pathogenesis of T2D.

Integrative HPLC profiling and transcriptome analysis revealed insights into anthocyanin accumulation and key genes at three developmental stages of black rice (Oryza sativa. L) caryopsis.

Anthocyanins are plants' secondary metabolites belonging to the flavonoid class with potential health-promoting properties. They are greatly employed in the food industry as natural alternative food colorants for dairy and ready-to-eat desserts and pH indicators. These tremendous advantages make them economically important with increasing market trends. Black rice is a rich source of anthocyanin that can be used to ensure food and nutritional security around the world. However, research on anthocyanin accumulation and gene expression during rice caryopsis development is lacking. In this study, we combined high-performance liquid chromatography (HPLC) and transcriptome analysis to profile the changes in anthocyanin content and gene expression dynamics at three developmental stages (milky, doughy, and mature). Our results showed that anthocyanin accumulation started to be visible seven days after flowering (DAF), increased rapidly from milky (11 DAF) to dough stage, then started decreasing after the peak was attained at 18 DAF. RNA-seq showed that 519 out of 14889, 477 out of 17914, and 1614 out of 18810 genes were uniquely expressed in the milky, doughy, and mature stages, respectively. We performed three pairwise comparisons: milky vs. dough, milky vs. mature, and dough vs. mature, and identified 6753, 9540, and 2531 DEGs, respectively. The DEGs' abundance was higher in milky vs. mature, with 5527 up-regulated genes and 4013 down-regulated genes, while it was smaller in dough vs. mature, with 1419 up-regulated genes and 1112 down-regulated DEGs. This result was consistent with the changes in anthocyanin profiling, and the expression of structural, regulatory, and transporter genes involved in anthocyanin biosynthesis showed their highest expression at the dough stage. Through the gene expression profile and protein interaction network, we deciphered six main contributors of the anthocyanin peak observed at dough stage, including OsANS, OsDFR, OsGSTU34, OsMYB3, OsbHLH015, and OsWD40-50. This study is the first to report the investigation of anthocyanin and gene expression at three developmental stages of black rice caryopsis. The findings of this study could aid in predicting the best harvesting time to achieve maximum anthocyanin content and the best time to collect samples for various gene expression analysis, laying the groundwork for future research into the molecular mechanisms underlying rice caryopsis coloration.

Role of caveolin-1 in metabolic programming of fetal brain

Mice lacking caveolin-1 (Cav1), a key protein of plasma membrane, exhibit brain aging at an early adult stage. Here, integrative analyses of metabolomics, transcriptomics, epigenetics, and single-cell data were performed to test the hypothesis that metabolic deregulation of fetal brain due to the ablation of Cav1 is linked to brain aging in these mice. The results of this study show that lack of Cav1 caused deregulation in the lipid and amino acid metabolism in the fetal brain, and genes associated with these deregulated metabolites were significantly altered in the brain upon aging. Moreover, ablation of Cav1 deregulated several metabolic genes in specific cell types of the fetal brain and impacted DNA methylation of those genes in coordination with mouse epigenetic clock. The findings of this study suggest that the aging program of brain is confounded by metabolic abnormalities in the fetal stage due to the absence of Cav1.

Peripheral immune landscape for hypercytokinemia in myasthenic crisis utilizing single-cell transcriptomics

BackgroundMyasthenia gravis (MG) is the most prevalent autoimmune disorder affecting the neuromuscular junction. A rapid deterioration in respiratory muscle can lead to a myasthenic crisis (MC), which represents a life-threatening condition with high mortality in MG. Multiple CD4+ T subsets and hypercytokinemia have been identified in the peripheral pro-inflammatory milieu during the crisis. However, the pathogenesis is complicated due to the many types of cells involved, leaving the underlying mechanism largely unexplored.MethodsWe conducted single-cell transcriptomic and immune repertoire sequencing on 33,577 peripheral blood mononuclear cells (PBMCs) from two acetylcholine receptor antibody-positive (AChR +) MG patients during MC and again three months post-MC. We followed the Scanpy workflow for quality control, dimension reduction, and clustering of the single-cell data. Subsequently, we annotated high-resolution cell types utilizing transfer-learning models derived from publicly available single-cell immune datasets. RNA velocity calculations from unspliced and spliced mRNAs were applied to infer cellular state progression. We analyzed cell communication and MG-relevant cytokines and chemokines to identify potential inflammation initiators.ResultsWe identified a unique subset of monocytes, termed monocytes 3 (FCGR3B+ monocytes), which exhibited significant differential expression of pro-inflammatory signaling pathways during and after the crisis. In line with the activated innate immune state indicated by MC, a high neutrophil–lymphocyte ratio (NLR) was confirmed in an additional 22 AChR + MC patients in subsequent hemogram analysis and was associated with MG-relevant clinical scores. Furthermore, oligoclonal expansions were identified in age-associated B cells exhibiting high autoimmune activity, and in CD4+ and CD8+ T cells demonstrating persistent T exhaustion.ConclusionsIn summary, our integrated analysis of single-cell transcriptomics and TCR/BCR sequencing has underscored the role of innate immune activation which is associated with hypercytokinemia in MC. The identification of a specific monocyte cluster that dominates the peripheral immune profile may provide some hints into the etiology and pathology of MC. However, future functional studies are required to explore causality.