Integrated Transcriptome Analysis Research Articles

Integrative analysis of transcriptome, DNA methylome and chromatin accessibility reveals candidate therapeutic targets in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and is characterized by primary left ventricular hypertrophy usually caused by mutations in sarcomere genes. The mechanism underlying cardiac remodeling in HCM remains incompletely understood. An investigation of HCM through integrative analysis at multi-omics levels will be helpful for treating HCM. DNA methylation and chromatin accessibility, as well as gene expression, were assessed by nucleosome occupancy and methylome sequencing (NOMe-seq) and RNA-seq, respectively, using the cardiac tissues of HCM patients. Compared with those of the controls, the transcriptome, DNA methylome and chromatin accessibility of the HCM myocardium showed multifaceted differences. At the transcriptome level, HCM hearts returned to the fetal gene program through decreased sarcomeric and metabolic gene expression and increased extracellular matrix gene expression. In the DNA methylome, hypermethylated and hypomethylated differentially methylated regions (DMRs) were identified in HCM. At the chromatin accessibility level, HCM hearts showed changes in different genome elements. Several transcription factors (TFs), including SP1 and EGR1, exhibited a fetal-like pattern of binding motifs in nucleosome-depleted regions (NDRs) in HCM. In particular, the inhibition of SP1 or EGR1 in an HCM mouse model harboring sarcomere mutations markedly alleviated the HCM phenotype of the mutant mice and reversed fetal gene reprogramming. Overall, this study not only provides a high precision multi-omics map of HCM heart tissue but also sheds light on the therapeutic strategy by intervening the fetal gene reprogramming in HCM.

Whole genome and transcriptome integrated analyses guide clinical care of pediatric poor prognosis cancers

The role for routine whole genome and transcriptome analysis (WGTA) for poor prognosis pediatric cancers remains undetermined. Here, we characterize somatic mutations, structural rearrangements, copy number variants, gene expression, immuno-profiles and germline cancer predisposition variants in children and adolescents with relapsed, refractory or poor prognosis malignancies who underwent somatic WGTA and matched germline sequencing. Seventy-nine participants with a median age at enrollment of 8.8 y (range 6 months to 21.2 y) are included. Germline pathogenic/likely pathogenic variants are identified in 12% of participants, of which 60% were not known prior. Therapeutically actionable variants are identified by targeted gene report and whole genome in 32% and 62% of participants, respectively, and increase to 96% after integrating transcriptome analyses. Thirty-two molecularly informed therapies are pursued in 28 participants with 54% achieving a clinical benefit rate; objective response or stable disease ≥6 months. Integrated WGTA identifies therapeutically actionable variants in almost all tumors and are directly translatable to clinical care of children with poor prognosis cancers.

Integrative analysis of transcriptome and target metabolites uncovering flavonoid biosynthesis regulation of changing petal colors in Nymphaea ‘Feitian 2’

BackgroundNymphaea (waterlily) is known for its rich colors and role as an important aquatic ornamental plant globally. Nymphaea atrans and some hybrids, including N. ‘Feitian 2,’ are more appealing due to the gradual color change of their petals at different flower developmental stages. The petals of N. ‘Feitian 2’ gradually change color from light blue-purple to deep rose-red throughout flowering. The mechanism of the phenomenon remains unclear.ResultsIn this work, flavonoids in the petals of N. ‘Feitian 2’ at six flowering stages were examined to identify the influence of flavonoid components on flower color changes. Additionally, six cDNA libraries of N. ‘Feitian 2’ over two blooming stages were developed, and the transcriptome was sequenced to identify the molecular mechanism governing petal color changes. As a result, 18 flavonoid metabolites were identified, including five anthocyanins and 13 flavonols. Anthocyanin accumulation during flower development is the primary driver of petal color change. A total of 12 differentially expressed genes (DEGs) in the flavonoid biosynthesis pathway were uncovered, and these DEGs were significantly positively correlated with anthocyanin accumulation. Six structural genes were ultimately focused on, as their expression levels varied significantly across different flowering stages. Moreover, 104 differentially expressed transcription factors (TFs) were uncovered, and three MYBs associated with flavonoid biosynthesis were screened. The RT-qPCR results were generally aligned with high-throughput sequencing results.ConclusionsThis research offers a foundation to clarify the mechanisms underlying changes in the petal color of waterlilies.

Response analysis of Pinus sibirica to pine wood nematode infection through transcriptomics and metabolomics study.

Pinus sibirica is primarily distributed in Siberia. Owing to its excellent cold resistance and development potential, it has become an important introduced tree species in the Greater Xing'an area of China. Pine wilt disease, triggered by the pine wood nematode (PWN, Bursaphelenchus xylophilus), constitutes a profoundly critical affliction within forest ecosystems. Its incidence has extended to the northeastern region of China in recent years. To explore the potential host status of P. sibirica in the Greater Xing'an area for PWN and to elucidate the responses following inoculation, artificial inoculation, transcriptomics, and metabolomics methods were used. In the artificial inoculation experiments, quantitative analysis of nematode populations within the trees demonstrated that PWN exhibited normal growth and reproductive capabilities within P. sibirica. Subsequently, transcriptome and metabolome sequencing were conducted at four time points before disease onset (3-, 5-, 7-, and 9-days post inoculation). Gene trend analysis and differentially expressed gene screening were employed and the results indicated that genes associated with the flavonoid biosynthesis pathway exhibited predominant enrichment among the up-regulated genes. Metabolome analysis showed that the abundance of flavonoid-related metabolites in P. sibirica increased after inoculation with PWN. Integrated analysis of transcriptome and metabolome revealed that after PWN inoculation in P. sibirica, two chalcone synthase (chs) genes and a chalcone isomerase (chi) gene were significantly upregulated, and the upregulation should accumulate naringenin, pinocembrin, and apigenin to help P. sibirica resist infection of PWN. The results suggested that flavonoid biosynthesis pathway continued to respond after P. sibirica was infected with PWN and played an important role in the interaction between P. sibirica and PWN.

Integrative transcriptome analysis uncovers common components containing CPS2 regulated by maize lncRNA GARR2 in gibberellin response.

The combined transcriptome outcome provides an important clue to the regulatory cascade centering on lncRNA GARR2 and CPS2 gene in GA response. Long non-coding RNAs (lncRNAs) serve as regulatory components in transcriptional hierarchy governing multiple aspects of biological processes. Dissecting regulatory mechanisms underpinning tetracyclic diterpenoid gibberellin (GA) cascade holds both theoretical and applied significance. However, roles of lncRNAs in transcriptional modulation of GA pathway remain largely elusive. Gypsy retrotransposon-derived GIBBERELLIN RESPONSIVE lncRNA2 (GARR2) has been reported as GA-responsive maize lncRNA. Here a novel GARR2-edited line garr2-1 was identified, characteristic of GA-induced phenotype of increased seedling height and elongated leaf sheath. Transcriptome analysis indicated that transcriptional abundance of five genes [ent-copalyl diphosphate synthase2 (CPS2), ent-kaurene synthase4 (KS4), ent-kaurene synthase6 (KS6), ent-kaurene oxidase2 (KO2), and ent-kaurenoic acid oxidase1/Dwarf3 (KAO1/D3)] was elevated in garr2-1 for early steps of GA biosynthesis. Five GA biosynthetic genes as hub regulators were interlaced to shape regulatory network of GA response. Different transcriptome resources were integrated to discover common differentially expressed genes (DEGs) in the independent GARR2-edited lines GARR2KO and garr2-1. A total of 320 common DEGs were retrieved. These common DEGs were enriched in diterpenoid biosynthetic pathway. Integrative transcriptome analysis revealed the common CPS2 encoding the CPS enzyme that catalyzes the conversion of the precursor trans-geranylgeranyl diphosphate to ent-copalyl diphosphate. The up-regulated CPS2 supported the GA-induced phenotype of slender seedlings observed in the independent GARR2-edited lines GARR2KO and garr2-1. Our integrative transcriptome analysis uncovers common components of the GA pathway regulated by lncRNA GARR2. These common components, especially for the GA biosynthetic gene CPS2, provide a valuable resource for further delineating the underlying mechanisms of lncRNA GARR2 in GA response.

Integrative analyses of bulk and single-cell transcriptomics reveals the infiltration and crosstalk of cancer-associated fibroblasts as a novel predictor for prognosis and microenvironment remodeling in intrahepatic cholangiocarcinoma

BackgroundIntrahepatic cholangiocarcinoma (ICC) is a highly malignant neoplasm and characterized by desmoplastic matrix. The heterogeneity and crosstalk of tumor microenvironment remain incompletely understood.MethodsTo address this gap, we performed Weighted Gene Co-expression Network Analysis (WGCNA) to identify and construct a cancer associated fibroblasts (CAFs) infiltration biomarker. We also depicted the intercellular communication network and important receptor-ligand complexes using the single-cell transcriptomics analysis of tumor and Adjacent normal tissue.ResultsThrough the intersection of TCGA DEGs and WGCNA module genes, 784 differential genes related to CAFs infiltration were obtained. After a series of regression analyses, the CAFs score was generated by integrating the expressions of EVA1A, APBA2, LRRTM4, GOLGA8M, BPIFB2, and their corresponding coefficients. In the TCGA-CHOL, GSE89748, and 107,943 cohorts, the high CAFs score group showed unfavorable survival prognosis (p < 0.001, p = 0.0074, p = 0.028, respectively). Additionally, a series of drugs have been predicted to be more sensitive to the high-risk group (p < 0.05). Subsequent to dimension reduction and clustering, thirteen clusters were identified to construct the single-cell atlas. Cell-cell interaction analysis unveiled significant enhancement of signal transduction in tumor tissues, particularly from fibroblasts to malignant cells via diverse pathways. Moreover, SCENIC analysis indicated that HOXA5, WT1, and LHX2 are fibroblast specific motifs.ConclusionsThis study reveals the key role of fibroblasts - oncocytes interaction in the remodeling of the immunosuppressive microenvironment in intrahepatic cholangiocarcinoma. Subsequently, it may trigger cascade activation of downstream signaling pathways such as PI3K-AKT and Notch in tumor, thus initiating tumorigenesis. Targeted drugs aimed at disrupting fibroblasts-tumor cell interaction, along with associated enrichment pathways, show potential in mitigating the immunosuppressive microenvironment that facilitates tumor progression.

Abstract PO5-06-12: Genetic screen identifies epigenetic drivers of breast cancer brain metastasis

Abstract Metastatic breast cancer remains a major cause of cancer-related deaths in women, and there are few effective therapies against this advanced disease. Brain metastases, found in 15~35% of patients with breast cancer, are often incurable. Accumulating evidence suggests that key steps of tumor progression and metastasis are controlled by reversible epigenetic mechanisms. To systematically identify the druggable epigenetic regulators essential for breast cancer brain metastasis, we conducted an in vivo shRNA screen and identified multiple epigenetic regulators, including RNA acetyltransferase NAT10 as drivers of breast cancer brain metastasis. Knockdown of NAT10 significantly restrains breast cancer cell proliferation and migration in vitro and brain metastases in vivo. NAT10 promotes breast cancer cell growth through its RNA helicase activity but not its acetyltransferase activity. Integrative analyses of transcriptomics, proteomics, and NAT10 eCLIP-Seq data identify NAT10 downstream targets critical for breast cancer progression and metastasis. Thus, these results reveal novel therapeutic strategies to treat metastatic breast cancer. Citation Format: Jocelyn Chen, Wesley Cai, Peng Xu, Don Nguyen, Qin Yan. Genetic screen identifies epigenetic drivers of breast cancer brain metastasis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-12.

Abstract PO1-15-02: Comprehensive characterization of genetic interactions in breast cancer reveals therapeutic vulnerabilities

Abstract Background: Genome-informed and genome-targeted precision treatment for breast cancer have achieved remarkable progress in improving clinical outcomes for patients with specific genetic alterations. However, treatment efficacy is compromised by the current practice of basing treatment decision-making solely on single driver alterations, without considering the role of genetic interactions. Consequently, it is of great necessity to conduct systematic investigations to determine the clinical relevance of genetic interactions. Methods: We established a large-scale multi-omics cohort (N=873) and a real-world clinical sequencing cohort (N=4,421) representing the Asian breast cancer population. Detailed treatment records were collected. We then investigated the prognostic and predictive effects of genetic interactions based on multivariate Cox proportional hazards model and logistic regression model. To validate our findings, we utilized patient-derived organoids and tumor fragments to confirm the associations observed between genetic interactions and drug response. Results: Through integrated analysis of genomics, transcriptomics, proteomics, and metabolomics, we constructed a network comprising 54 co-occurring events and 38 mutually exclusive events, elucidating their association with dysregulated biological processes. External validations were performed in TCGA-BRCA, MSK-IMPACT, METABRIC, and AACR-GENIE datasets, respectively. Furthermore, we systematically revealed the prognostic effects of genetic interactions across distinct clinical subtypes. In triple-negative breast cancer, we found that the co-occurrence of PIK3CAmut-FOXA1mut was associated with unfavorable distant metastasis-free survival while TP53mut-MYBamp and TP53mut-CCNE1amp were associated with decreased overall survival. Additionally, we characterized the genetic interactions that impact the clinical outcomes of patients undergoing specific treatments in the neoadjuvant, adjuvant, and advanced settings. Notably, we identified associations such as TP53mut-AURKAamp with tamoxifen resistance, ERBB2amp-PAK1amp with resistance to trastuzumab-pertuzumab combinations, germline BRCA1mut-MYCamp with sensitivity to PARP1 inhibitors, and TP53mut-MYBamp with immunotherapy resistance. Conclusion: Overall, the consideration of genetic interactions may enhance our understanding of the heterogeneity in treatment response and complement ongoing efforts in precision oncology. Our study suggests that decision-making regarding genome-informed and genome-targeted treatment should extend beyond the scope of single driver alterations. Citation Format: Caijin Lin, Xi Jin, Ding Ma, Chao Chen, Xin Hu, Yi-Zhou Jiang, Zhi-Ming Shao. Comprehensive characterization of genetic interactions in breast cancer reveals therapeutic vulnerabilities [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-02.

An integrated analysis of transcriptome and metabolome to reveal the effects of temperature stress on energy metabolism and physiological responses in Schizothorax wangchiachii muscles

The unique hydrological situation of Jinsha River and the construction and operation of large hydropower stations may cause resident Schizothorax wangchiachii to experience acute changes in water temperature. To reveal the response mechanisms of S. wangchiachii to high and low temperature stress, we conducted a stress experiment. Compared with the control group, the activity of antioxidant enzymes (SOD\\CAT) increased first and then decreased at high temperature, while MDA activity in the low temperature group continued to increase slowly. The activities of glycolytic enzymes were increased initially and then decreased including HK and PFK, but PK activity continued to increase throughout the stress period. Transcriptome sequencing showed that a large number of genes in muscle tissues were differentially expressed after high and low temperature stress, and the main enriched pathways included glycosaminoglycan biosynthesis, lysine degradation, glycerolipid metabolism, the PPAR signaling pathway, and the adipocytokine signaling pathway. Significant metabolic pathways were identified as enriched according to the metabolic differences,such as glycerophospholipid metabolism, qRT-PCR was used to verify gene expression. The results suggested that both high temperature and low temperature stress induced physiological responses in S. wangchiachii that maintained homeostasis by regulating glucolipid metabolism and amino acid degradation. This study revealed the response mechanisms of S. wangchiachii to high and low temperature stress, information that may serve as a reference for the study of responses in other fish to environmental changes in the upper reaches of the Yangtze River or where they are displaced by hydropower station construction.

Identification of pathogenic-like fungal species on Akebia trifoliata fruit by integrating transcriptome analysis and specific sequences of putative pathogens

Large-scale cultivation of recently domesticated crops poses great potential disease risk, so rapid and comprehensive identification of pathogenic microbes is of great significance and value for the commercial development of wild plant resources. Akebia trifoliata is widely cultivated in various regions of China, although its domestication remains in the early stages. Here, to avoid large economic losses, we identified the pathogenic-like fungi of widely cultivated A. trifoliata based on causal relationship between pathogens and hosts such as discernible symptoms, fungal diversity and differentially expressed sequences in symptomatic and asymptomatic fruits. First, we observed partly discernible symptoms of four diseases (powdery mildew, anthracnose, fruit shrinkage and brown spot) by observing disease development in the field. Second, comparative analysis of the ITS sequences between the symptomatic and asymptomatic rind bulks suggested that Cercospora, Colletotrichum and Strelitziana could contain pathogenic-like fungi. Finally, interaction transcriptome sequencing afforded strong evidence for 20 pathogenic-like fungi associated with five reported diseases in A. trifoliata and other plants and seven putative unknown diseases, among which the pathogenic-like fungi causing anthracnose, powdery mildew and brown spot were the three top richness fungi in symptomatic fruit rind. This study provides a new strategy for the rapid identification of pathogenic-like fungi in plants and provides some reference for farmers to prevent and control A. trifoliata diseases.

Investigation of heat stress responses and adaptation mechanisms by integrative metabolome and transcriptome analysis in tea plants (Camellia sinensis).

Extreme high temperature has deleterious impact on the yield and quality of tea production, which has aroused the attention of growers and breeders. However, the mechanisms by which tea plant varieties respond to extreme environmental heat is not clear. In this study, we analyzed physiological indices, metabolites and transcriptome differences in three different heat-tolerant tea plant F1 hybrid progenies. Results showed that the antioxidant enzyme activity, proline, and malondialdehyde were significantly decreased in heat-sensitive 'FWS' variety, and the accumulation of reactive oxygen molecules such as H2O2 and O2- was remarkably increased during heat stress. Metabolomic analysis was used to investigate the metabolite accumulation pattern of different varieties in response to heat stress. The result showed that a total of 810 metabolites were identified and more than 300 metabolites were differentially accumulated. Transcriptional profiling of three tea varieties found that such genes encoding proteins with chaperon domains were preferentially expressed in heat-tolerant varieties under heat stress, including universal stress protein (USP32, USP-like), chaperonin-like protein 2 (CLP2), small heat shock protein (HSP18.1), and late embryogenesis abundant protein (LEA5). Combining metabolomic with transcriptomic analyses discovered that the flavonoids biosynthesis pathway was affected by heat stress and most flavonols were up-regulated in heat-tolerant varieties, which owe to the preferential expression of key FLS genes controlling flavonol biosynthesis. Take together, molecular chaperons, or chaperon-like proteins, flavonols accumulation collaboratively contributed to the heat stress adaptation in tea plant. The present study elucidated the differences in metabolite accumulation and gene expression patterns among three different heat-tolerant tea varieties under extreme ambient high temperatures, which helps to reveal the regulatory mechanisms of tea plant adaptation to heat stress, and provides a reference for the breeding of heat-tolerant tea plant varieties.

489. Deciphering the Molecular Basis of Aging in Substance Use Disorder: Integrative Transcriptomic and Methylomic Analysis

489. Deciphering the Molecular Basis of Aging in Substance Use Disorder: Integrative Transcriptomic and Methylomic Analysis

Gender-specific effects of polystyrene nanoplastic exposure on triclosan-induced reproductive toxicity in zebrafish (Danio rerio)

Nanoplastics (NPs) and triclosan (TCS) are ubiquitous emerging environmental contaminants detected in human samples. While the reproductive toxicity of TCS alone has been studied, its combined effects with NPs remain unclear. Herein, we employed Fourier transform infrared spectroscopy and dynamic light scattering to characterize the coexposure of polystyrene nanoplastics (PS-NPs, 50 nm) with TCS. Then, adult zebrafish were exposed to TCS at environmentally relevant concentrations (0.361–48.2 μg/L), with or without PS-NPs (1.0 mg/L) for 21 days. TCS biodistribution in zebrafish tissues was investigated using ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry. Reproductive toxicity was assessed through gonadal histopathology, fertility tests, changes in steroid hormone synthesis and gene expression within the hypothalamus-pituitary-gonad-liver (HPGL) axis. Transcriptomics and proteomics were applied to explore the underlying mechanisms. The results showed that PS-NPs could adsorb TCS, thus altering the PS-NPs' physical characteristics. Our observations revealed that coexposure with PS-NPs reduced TCS levels in the ovaries, livers, and brains of female zebrafish. Conversely, in males, coexposure with PS-NPs increased TCS levels in the testes and livers, while decreasing them in the brain. We found that co-exposure mitigated TCS-induced ovary development inhibition while exacerbated TCS-induced spermatogenesis suppression, resulting in increased embryonic mortality and larval malformations. This co-exposure influenced the expression of genes linked to steroid hormone synthesis (cyp11a1, hsd17β, cyp19a1) and attenuated the TCS-decreased estradiol (E2) in females. Conversely, testosterone levels were suppressed, and E2 levels were elevated due to the upregulation of specific genes (cyp11a1, hsd3β, cyp19a1) in males. Finally, the integrated analysis of transcriptomics and proteomics suggested that the aqp12-dctn2 pathway was involved in PS-NPs' attenuation of TCS-induced reproductive toxicity in females, while the pck2-katnal1 pathway played a role in PS-NPs' exacerbation of TCS-induced reproductive toxicity in males. Collectively, PS-NPs altered TCS-induced reproductive toxicity by disrupting the HPGL axis, with gender-specific effects.

Integrative proteomic and transcriptomic analysis in the female goat ovary to explore the onset of puberty

Puberty is considered a prerequisite for affecting reproductive performance and productivity. Little was known about molecular changes in pubertal goat ovaries. Therefore, we measured and performed a correlation analysis of the mRNA and proteins changes in the pre-pubertal and pubertal goat ovaries. The results showed that only six differentially expressed genes and differentially abundant proteins out of 18,139 genes and 7550 proteins quantified had significant correlations. CNTN2 and THBS1, discovered in the mRNA-mRNA interaction network, probably participated in pubertal and reproductive regulation by influencing GnRH receptor signals, follicular development, and ovulation. The predicted core transcription factors may either promote or inhibit the expression of reproductive genes and act synergistically to maintain normal reproductive function in animals. The interaction between PKM and TIMP3 with other proteins may impact animal puberty through energy metabolism and ovarian hormone secretion. Pathway enrichment analyses revealed that the co-associated key pathways between ovarian genes and proteins at puberty included calcium signalling pathway and olfactory transduction. These pathways were associated with gonadotropin-releasing hormone synthesis and secretion, signal transmission, and cell proliferation. In summary, these results enriched the potential molecules and signalling pathways that affect puberty and provided new insights for regulating and promoting the onset of puberty. SignificanceThis study conducted the first transcriptomic and proteomic correlation analysis of pre-pubertal and pubertal goat ovaries and identified six significantly correlated molecules at both the gene and protein levels. Meanwhile, we were drawn to several molecules and signalling pathways that may play a regulatory role in the onset of puberty and reproduction by influencing reproductive-related gene expression, GnRH receptor signals, energy metabolism, ovarian hormone secretion, follicular development, and ovulation. This information contributed to identify potential biomarkers in pubertal goat ovaries, which was vital for predicting the onset of puberty and improving livestock performance.

Integrated Transcriptomics and Metabolomics Analysis Reveal the Regulatory Mechanisms Underlying Sodium Butyrate-Induced Carotenoid Biosynthesis in Rhodotorula glutinis.

Sodium butyrate (SB) is a histone deacetylase inhibitor that can induce changes in gene expression and secondary metabolite titers by inhibiting histone deacetylation. Our preliminary analysis also indicated that SB significantly enhanced the biosynthesis of carotenoids in the Rhodotorula glutinis strain YM25079, although the underlying regulatory mechanisms remained unclear. Based on an integrated analysis of transcriptomics and metabolomics, this study revealed changes in cell membrane stability, DNA and protein methylation levels, amino acid metabolism, and oxidative stress in the strain YM25079 under SB exposure. Among them, the upregulation of oxidative stress may be a contributing factor for the increase in carotenoid biosynthesis, subsequently enhancing the strain resistance to oxidative stress and maintaining the membrane fluidity and function for normal cell growth. To summarize, our results showed that SB promoted carotenoid synthesis in the Rhodotorula glutinis strain YM25079 and increased the levels of the key metabolites and regulators involved in the stress response of yeast cells. Additionally, epigenetic modifiers were applied to produce fungal carotenoid, providing a novel and promising strategy for the biosynthesis of yeast-based carotenoids.

Integrative analysis of metabolomics and transcriptomics to uncover biomarkers in sepsis

To utilize metabolomics in conjunction with RNA sequencing to identify biomarkers in the blood of sepsis patients and discover novel targets for diagnosing and treating sepsis. In January 2019 and December 2020, blood samples were collected from a cohort of 16 patients diagnosed with sepsis and 11 patients diagnosed with systemic inflammatory response syndrome (SIRS). Non-targeted metabolomics analysis was conducted using liquid chromatography coupled with mass spectrometry (LC–MS/MS technology), while gene sequencing was performed using RNA sequencing. Afterward, the metabolite data and sequencing data underwent quality control and difference analysis, with a fold change (FC) greater than or equal to 2 and a false discovery rate (FDR) less than 0.05.Co-analysis was then performed to identify differential factors with consistent expression trends based on the metabolic pathway context; KEGG enrichment analysis was performed on the crossover factors, and Meta-analysis of the targets was performed at the transcriptome level using the public dataset. In the end, a total of five samples of single nucleated cells from peripheral blood (two normal controls, one with systemic inflammatory response syndrome, and two with sepsis) were collected and examined to determine the cellular location of the essential genes using 10× single cell RNA sequencing (scRNA-seq). A total of 485 genes and 1083 metabolites were found to be differentially expressed in the sepsis group compared to the SIRS group. Among these, 40 genes were found to be differentially expressed in both the metabolome and transcriptome. Functional enrichment analysis revealed that these genes were primarily involved in biological processes related to inflammatory response, immune regulation, and amino acid metabolism. Furthermore, a meta-analysis identified four genes, namely ITGAM, CD44, C3AR1, and IL2RG, which were highly expressed in the sepsis group compared to the normal group (P < 0.05). Additionally, scRNA-seq analysis revealed that the core genes ITGAM and C3AR1 were predominantly localized within the macrophage lineage. The primary genes ITGAM and C3AR1 exhibit predominant expression in macrophages, which play a significant role in inflammatory and immune responses. Moreover, these genes show elevated expression levels in the plasma of individuals with sepsis, indicating their potential as valuable subjects for further research in sepsis.

Integrated metabolome and transcriptome analyses reveal the role of BoGSTF12 in anthocyanin accumulation in Chinese kale (Brassica oleracea var. alboglabra)

BackgroundThe vivid red, purple, and blue hues that are observed in a variety of plant fruits, flowers, and leaves are produced by anthocyanins, which are naturally occurring pigments produced by a series of biochemical processes occurring inside the plant cells. The purple-stalked Chinese kale, a popular vegetable that contains anthocyanins, has many health benefits but needs to be investigated further to identify the genes involved in the anthocyanin biosynthesis and translocation in this vegetable.ResultsIn this study, the purple- and green-stalked Chinese kale were examined using integrative transcriptome and metabolome analyses. The content of anthocyanins such as cyanidin-3-O-(6″-O-feruloyl) sophoroside-5-O-glucoside, cyanidin-3,5-O-diglucoside (cyanin), and cyanidin-3-O-(6″-O-p-hydroxybenzoyl) sophoroside-5-O-glucoside were considerably higher in purple-stalked Chinese kale than in its green-stalked relative. RNA-seq analysis indicated that 23 important anthocyanin biosynthesis genes, including 3 PAL, 2 C4H, 3 4CL, 3 CHS, 1 CHI, 1 F3H, 2 FLS, 2 F3’H, 1 DFR, 3 ANS, and 2 UFGT, along with the transcription factor BoMYB114, were significantly differentially expressed between the purple- and green-stalked varieties. Results of analyzing the expression levels of 11 genes involved in anthocyanin production using qRT-PCR further supported our findings. Association analysis between genes and metabolites revealed a strong correlation between BoGSTF12 and anthocyanin. We overexpressed BoGSTF12 in Arabidopsis thaliana tt19, an anthocyanin transport mutant, and this rescued the anthocyanin-loss phenotype in the stem and rosette leaves, indicating BoGSTF12 encodes an anthocyanin transporter that affects the accumulation of anthocyanins.ConclusionThis work represents a key step forward in our understanding of the molecular processes underlying anthocyanin production in Chinese kale. Our comprehensive metabolomic and transcriptome analyses provide important insights into the regulatory system that controls anthocyanin production and transport, while providing a foundation for further research to elucidate the physiological importance of the metabolites found in this nutritionally significant vegetable.

Integrated analysis of transcriptome and miRNAome reveals the heat stress response of Pinellia ternata seedlings

Pinellia ternata (Thunb.) Briet., a valuable herb native to China, is susceptible to the “sprout tumble” phenomenon because of high temperatures, resulting in a significant yield reduction. However, the molecular regulatory mechanisms underlying the response of P. ternata to heat stress are not well understood. In this study, we integrated transcriptome and miRNAome sequencing to identify heat-response genes, microRNAs (miRNAs), and key miRNA-target pairs in P. ternata that differed between heat-stress and room-temperature conditions. Transcriptome analysis revealed extensive reprogramming of 4,960 genes across various categories, predominantly associated with cellular and metabolic processes, responses to stimuli, biological regulation, cell parts, organelles, membranes, and catalytic and binding activities. miRNAome sequencing identified 1,597 known/conserved miRNAs that were differentially expressed between the two test conditions. According to the analysis, genes and miRNAs associated with the regulation of transcription, DNA template, transcription factor activity, and sequence-specific DNA binding pathways may play a major role in the resistance to heat stress in P. ternata. Integrated analysis of the transcriptome and miRNAome expression data revealed 41 high-confidence miRNA-mRNA pairs, forming 25 modules. MYB-like proteins and calcium-responsive transcription coactivators may play an integral role in heat-stress resistance in P. ternata. Additionally, the candidate genes and miRNAs were subjected to quantitative real-time polymerase chain reaction to validate their expression patterns. These results offer a foundation for future studies exploring the mechanisms and critical genes involved in heat-stress resistance in P. ternata.

From genome to proteome: Comprehensive identification of venom toxins from the Chinese funnel-web spider (Macrothelidae: Macrothele yani)

Macrothelidae is a family of mygalomorph spiders containing the extant genera Macrothele and Vacrothele. China is an important center of diversity for Macrothele with 65 % of the known species occurring there. Previous work on Macrothele was able to uncover several important toxin compounds including Raventoxin which may have applications in biomedicine and agricultural chemistry. Despite the importance of Macrothele spiders, high-quality reference genomes are still lacking, which hinders our understanding and application of the toxin compounds. In this study, we assembled the genome of the Macrothele yani to help fill gaps in our understanding of toxin biology in this lineage of spiders to encourage the future study and applications of these compounds. The final assembled genome was 6.79 Gb in total length, had a contig N50 of 21.44 Mb, and scaffold N50 of 156.16 Mb. Hi-C scaffolding assigned 98.19 % of the genome to 46 pseudo-chromosomes with a BUSCO score of 95.7 % for the core eukaryotic gene set. The assembled genome was found to contain 75.62 % repetitive DNA and a total of 39,687 protein-coding genes were annotated making it the spider genome with highest number of genes. Through integrated analysis of venom gland transcriptomics and venom proteomics, a total of 194 venom toxins were identified, including 38 disulfide-rich peptide neurotoxins, among which 12 were ICK knottin peptides. In summary, we present the first high-quality genome assembly at the chromosomal level for any Macrothelidae spider, filling an important gap in our knowledge of these spiders. Such high-quality genomic data will be invaluable as a reference in resolving Araneae spider phylogenies and in screening different spider species for novel compounds applicable to numerous medical and agricultural applications.

Integrative single-cell chromatin and transcriptome analysis of human plasma cell differentiation

AbstractPlasma cells (PCs) are highly specialized cells representing the end stage of B-cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drive the early stages of PC differentiation. We combined single cell (sc) RNA sequencing (RNA-seq) and scATAC-seq to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to the AP-1 transcription factor family that may represent key transcriptional nodes involved in PC differentiation. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts had already undergone epigenetic remodeling related to PC profile together with unfolded protein response activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature PC maturation and biological functions. These data thus provide critical insights into epigenetic- and transcription–mediated reprogramming events that sustain PC differentiation.