Enriched Transcription Factor Research Articles

Intestinal carbapenem-resistant Klebsiella pneumoniae undergoes complex transcriptional reprogramming following immune activation

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.

Rummagene: massive mining of gene sets from supporting materials of biomedical research publications

Many biomedical research publications contain gene sets in their supporting tables, and these sets are currently not available for search and reuse. By crawling PubMed Central, the Rummagene server provides access to hundreds of thousands of such mammalian gene sets. So far, we scanned 5,448,589 articles to find 121,237 articles that contain 642,389 gene sets. These sets are served for enrichment analysis, free text, and table title search. Investigating statistical patterns within the Rummagene database, we demonstrate that Rummagene can be used for transcription factor and kinase enrichment analyses, and for gene function predictions. By combining gene set similarity with abstract similarity, Rummagene can find surprising relationships between biological processes, concepts, and named entities. Overall, Rummagene brings to surface the ability to search a massive collection of published biomedical datasets that are currently buried and inaccessible. The Rummagene web application is available at https://rummagene.com.

Abstract 7346: Optimizing pan-cancer driver pathway analysis from patient transcriptomics and phosphoproteomics

Abstract Alterations in cell signaling pathways drive cancer progression. Such changes can be detected by transcriptomics and proteomics profiling of tumor samples. Although proteomics profiling captures these alterations more directly, RNA-seq transcriptomics remains the most widely available and cost-effective source of data for elucidating tumor-specific cell signaling mechanisms. The eXpression2Kinases (X2K) pipeline is a computational workflow that starts with differentially expressed mRNAs, the identified differentially expressed genes are then used as input for transcription factor enrichment analysis. Then, the top-ranked transcription factors are connected via additional protein-protein interactions from several databases. Finally, the top-ranked transcription factors and the proteins that directly interact and connect these factors are subjected to kinase enrichment analysis. Such analysis identifies protein kinases most likely responsible for the observed changes in gene expression. The X2K pipeline can be applied to analyze tumor-specific transcriptomics to infer upstream regulatory transcription factors, protein intermediates, and kinases that are likely the drivers of cancer progression. While the X2K pipeline infers regulatory pathways from transcriptomics alone, phosphoproteomics can be used to independently validate the inferred kinases, and to calibrate the parameters of the X2K pipeline. Furthermore, this multiomics approach predicts cell signaling pathways with multiple layers of evidence by linking changes observed in the phosphoproteome to changes observed in the transcriptome. Here, we used the transcriptomics and phosphoproteomics profiles from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) pan-cancer dataset, including 1,020 samples across 10 different cancer types, to optimize the parameters of the X2K pipeline and predict pan-cancer as well as tumor-specific signaling pathways. While inferred pathways were distinct between different tumor types, we observed global enrichment of receptor tyrosine kinases and cell cycle kinases. These pathways were subsequently tested for association with patient survival to identify pathways that may be effective therapeutic targets, for example, PTK2 signaling was identified for many lung cancer tumors (LUAD and LSCC) as well as some breast, head & neck squamous. Altogether, the X2K pipeline presents a rational approach to better identify driver regulatory mechanisms from patient-specific profiling of tumors with both transcriptomics and phosphoproteomics. Citation Format: Eden Z. Deng, Giacomo B. Marino, Daniel J. Clarke, Weiping Ma, Pei Wang, Avi Ma'ayan. Optimizing pan-cancer driver pathway analysis from patient transcriptomics and phosphoproteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7346.

Abstract 6108: Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor (TF) activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus (sn) ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=33), Kenyan (n=25), and European American (EA, n=24) women in relation to genetic ancestry, risk factor exposures, clinical characteristics, and 5-year survival. To achieve this, we successfully isolated intact, high-quality single nuclei from archival frozen breast tumor tissue through an optimized combination of enzymatic digestion and automated tissue homogenization. We performed snMultiome sequencing of 82 tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Harmony), our dataset includes a total of 296,557 nuclei. Cancerous (163,419 nuclei) and non-cancerous (133,138 nuclei) cells were distinguished based on DNA copy number (CopyKat). Within the microenvironment, 11 major immune, epithelial, and stromal cell types were successfully annotated, exhibiting distinct patterns by population group (e.g. AA tumors showed markedly increased abundance of myeloid and T-cells, while Kenyan tumors showed increased abundance of pericytes and fibroblasts). A large number of enriched TFs within each cell type varied significantly by population group, suggesting distinct chromatin accessibility patterns related to genetic ancestry. Within cancerous cells, striking intra- and inter-tumoral heterogeneity was observed by genetic ancestry even within molecular subtype groups. Current efforts focus on in-depth molecular characterization of ancestry- and risk factor-related differences in the tumor epithelium and microenvironment and distinct signatures present in lethal disease. This project holds the potential to yield crucial insights into how ancestry or other factors may influence the etiology of different breast cancer subtypes, as well as produce clinically actionable biomarkers and therapeutic targets to enhance precision medicine within patient populations at high risk for aggressive disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany Jenkins-Lord, Tiffany H. Dorsey, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6108.

Diverging cell wall strategies for drought adaptation in two maize inbreds with contrasting lodging resistance.

The plant cell wall is a plastic structure of variable composition that constitutes the first line of defence against environmental challenges. Lodging and drought are two stressful conditions that severely impact maize yield. In a previous work, we characterised the cell walls of two maize inbreds, EA2024 (susceptible) and B73 (resistant) to stalk lodging. Here, we show that drought induces distinct phenotypical, physiological, cell wall, and transcriptional changes in the two inbreds, with B73 exhibiting lower tolerance to this stress than EA2024. In control conditions, EA2024 stalks had higher levels of cellulose, uronic acids and p-coumarate than B73. However, upon drought EA2024 displayed increased levels of arabinose-enriched polymers, such as pectin-arabinans and arabinogalactan proteins, and a decreased lignin content. By contrast, B73 displayed a deeper rearrangement of cell walls upon drought, including modifications in lignin composition (increased S subunits and S/G ratio; decreased H subunits) and an increase of uronic acids. Drought induced more substantial changes in gene expression in B73 compared to EA2024, particularly in cell wall-related genes, that were modulated in an inbred-specific manner. Transcription factor enrichment assays unveiled inbred-specific regulatory networks coordinating cell wall genes expression. Altogether, these findings reveal that B73 and EA2024 inbreds, with opposite stalk-lodging phenotypes, undertake different cell wall modification strategies in response to drought. We propose that the specific cell wall composition conferring lodging resistance to B73, compromises its cell wall plasticity, and renders this inbred more susceptible to drought.

Transposable elements mediate genetic effects altering the expression of nearby genes in colorectal cancer

Transposable elements (TEs) are prevalent repeats in the human genome, play a significant role in the regulome, and their disruption can contribute to tumorigenesis. However, TE influence on gene expression in cancer remains unclear. Here, we analyze 275 normal colon and 276 colorectal cancer samples from the SYSCOL cohort, discovering 10,231 and 5,199 TE-expression quantitative trait loci (eQTLs) in normal and tumor tissues, respectively, of which 376 are colorectal cancer specific eQTLs, likely due to methylation changes. Tumor-specific TE-eQTLs show greater enrichment of transcription factors, compared to shared TE-eQTLs suggesting specific regulation of their expression in tumor. Bayesian networks reveal 1,766 TEs as mediators of genetic effects, altering the expression of 1,558 genes, including 55 known cancer driver genes and show that tumor-specific TE-eQTLs trigger the driver capability of TEs. These insights expand our knowledge of cancer drivers, deepening our understanding of tumorigenesis and presenting potential avenues for therapeutic interventions.

Transcriptome analysis reveals the molecular mechanisms of rubber biosynthesis and laticifer differentiation during rubber seed germination.

The molecular mechanisms underlying the initiation of natural rubber synthesis and laticifer differentiation have not been fully elucidated. In this study, we conducted a time-series transcriptome analysis of five rubber tree tissues at four stages of seed germination. A total of 161,199 DEGs were identified between the two groups, including most 16,673 DEGs (A3 vs B3 and A3 vs C3) and lest 1,210 DEGs (C2 vs D2). We found that the maturation of the seed is accompanied by the formation of laticifer cells in cotyledon. Meanwhile, the analysis of hormones related genes expression may provide effective clues for us to promote the differentiation of laticifer cells in seeds by hormones in the future. In this study, hormone-related gene enrichment analyses revealed that IAA, GA, and CTK were activated in laticifer containing tissues. Similarly, GO and GEGG analysis showed that hormone pathways, especially the auxin pathway, are enriched. Gene expression clustering was analyzed using the short time-series expression miner (STEM), and the analysis revealed four distinct trends in the gene expression profiles. Moreover, we enriched transcription factor (TF) enrichment in cotyledon and embryonic axis tissues, and the MYB type exhibited the most significant difference. Furthermore, our findings revealed that genes related to rubber synthesis exhibited tissue-specific expression patterns during seed germination. Notably, key genes associated with rubber biosynthesis, specifically small rubber particle protein (SRPP) and cis-prenyltransferase (CPT), exhibited significant changes in expression in cotyledon and embryonic axis tissues, suggesting synchronous rubber synthesis with seed germination. Our staining results reveled that laticifer cells were exits in the cotyledon before seed imbibition stage. In conclusion, these results lay the foundation for exploring the molecular mechanisms underlying laticifer differentiation and rubber synthesis during seed germination, deepening our understanding of the initiation stages of rubber biosynthesis and laticifer differentiation.

P097 Spatially resolved insights into fistulating Crohn's disease pathogenesis: Unveiling molecular heterogeneity

Abstract Background Crohn's disease (CD) frequently results in fistula development in approximately 40% of patients due to sustained, transmural inflammation within the bowel wall. Despite advanced treatments, recurrence of fistulae affects one third of patients. Understanding its pathogenesis is crucial for targeted treatments, yet remains poorly defined. This study employs spatial transcriptomic and single-cell RNA-sequencing (scRNA-seq) technologies to characterise human fistulating CD tissue pathology. Methods Unbiased FFPE spatial transcriptomics (10x Visium) were applied to surgically resected, full-thickness (FT) tissue from 20 CD-associated fistulae cases and 15 controls. Subsequently, selected cases underwent further analysis using custom 500-plex MERFISH subcellular resolution spatial transcriptomics (Vizgen MERSCOPE). Additionally, a reference single-cell cohort encompassing patients with fistulating, stricturing, and inflammatory CD phenotypes, along with healthy controls, was generated. Optimisation of scRNA-seq for resected FT ileal tissue enabled the isolation and profiling of epithelial, immune, and stromal populations (10x Chromium). Computational analysis facilitated the spatial localisation of single-cell clusters enriched in fistula CD tissue, identifying key parameters linked to fistula development. Results The scRNA-seq data revealed diverse CD-specific cellular states adopted by intestinal fibroblasts, with IL11+ fibroblasts prominently expressing CD82, COL7A1, MMP1, CHI3L1, suggestive of pro-fibrotic signalling and regenerative morphogen pathways. This coincided with the expansion of a CD-specific subpopulation of pericytes (CCL19/CCL21) involved in leucocyte migration. Spatial profiling of CD fistula tracts by Visium and MERSCOPE unveiled an enrichment of key epithelial developmental transcription factors (e.g., GRHL3) and localised the pro-fibrotic signature displaying increased IL11 and MMP expression, along with perturbed WNT signalling within the fistula stroma. Active proliferation (HOPX, MKI67) was observed at the base of fistula tracts and within the stroma. Transcriptomic characterisation depicted a gradual loss in stem cell signature (LGR5, ASCL2, SMOC2) and supporting telocytes (POSTN) toward the leading edge of the fistula, signifying abnormal epithelium loss. Conclusion This pioneering study integrates multi-modal spatial transcriptomics and single-cell profiling to unveil the intricate cellular and molecular landscape in FT, fistulating CD pathology. Spatial analyses elucidate specific mechanisms involved in epithelial loss, stromal remodelling, and perturbed WNT signalling pathways within fistulating tissue, outlining a course of disrupted regenerative processes in fistulating CD.

SIRT7 sustains tumor development and radioresistance by repressing endoplasmic reticulum stress-induced apoptosis in cutaneous melanoma

Cutaneous melanoma is one of the most malignant human tumors and possesses strong resistance to radiotherapy. However, the mechanisms contribute to such radioresistance of melanoma is unclear. In this study, SIRT7 is identified to be higher-expressed in melanoma and positively correlated with melanoma staging. Under ionizing radiation (IR)-treatment condition, loss of SIRT7 compromised the survivability of melanoma cells showed by decreased proliferation, colony formation, migration, but enhancing apoptosis. Transcriptomic sequencing analysis indicated the apoptosis induced after SIRT7 knockdown is tightly related with the induction of endoplasmic reticulum stress (ER stress) by IR treatment. Loss of SIRT7 enhanced EIF2α acetylation and activated its phosphorylation to induce the expression of ER stress proteins including DDIT3, XBP1 and GRP78, among which DDIT3 is responsible for apoptosis induction. SIRT7 depletion enriched ER stress-activated transcription factor ATF4 at the promoter region of DDIT3 gene to transactivate its expression and induces apoptotic cascade in both mock- and IR-treatment conditions. Consistently, SIRT7 is highly upregulated in radioresistant melanoma cell strain and still modulates the ER-stress responsive genes to maintain the homeostasis of melanoma. Collectively, SIRT7 negatively regulates ER stress-activated apoptosis to enhance the survivability of melanoma cells in both non-IR- and IR-treatment conditions. Our study highlights the role of SIRT7 in repressing ER stress and the following apoptosis to sustain tumor development and mediate radioresistance in melanoma, which may suggest a novel intervention target for melanoma therapy.

Combined Transcriptomics and Metabolomics Identify Regulatory Mechanisms of Porcine Vertebral Chondrocyte Development In Vitro.

Porcine body length is closely related to meat production, growth, and reproductive performance, thus playing a key role in the profitability of the pork industry. Cartilage development is critical to longitudinal elongation of individual vertebrae. This study isolated primary porcine vertebral chondrocytes (PVCs) to clarify the complex mechanisms of elongation. We used transcriptome and target energy metabolome technologies to confirm crucial genes and metabolites in primary PVCs at different differentiation stages (0, 4, 8, and 12 days). Pairwise comparisons of the four stages identified 4566 differentially expressed genes (DEGs). Time-series gene cluster and functional analyses of these DEGs revealed four clusters related to metabolic processes, cartilage development, vascular development, and cell cycle regulation. We constructed a transcriptional regulatory network determining chondrocyte maturation. The network indicated that significantly enriched transcription factor (TF) families, including zf-C2H2, homeobox, TF_bZIP, and RHD, are important in cell cycle and differentiation processes. Further, dynamic network biomarker (DNB) analysis revealed that day 4 was the tipping point for chondrocyte development, consistent with morphological and metabolic changes. We found 24 DNB DEGs, including the TFs NFATC2 and SP7. Targeted energy metabolome analysis showed that most metabolites were elevated throughout chondrocyte development; notably, 16 differentially regulated metabolites (DRMs) were increased at three time points after cell differentiation. In conclusion, integrated metabolome and transcriptome analyses highlighted the importance of amino acid biosynthesis in chondrocyte development, with coordinated regulation of DEGs and DRMs promoting PVC differentiation via glucose oxidation. These findings reveal the regulatory mechanisms underlying PVC development and provide an important theoretical reference for improving pork production.

Cuproptosis/OXPHOS tendency prediction of prognosis and immune microenvironment of esophageal squamous cell carcinoma: Bioinformatics analysis and experimental validation

BackgroundCuproptosis is a newly discovered cell death mechanism that relies on mitochondrial respiration, for which oxidative phosphorylation (OXPHOS) is an essential part. However, the detailed mechanisms of cuproptosis associated with OXPHOS in esophageal squamous cell carcinoma (ESCC) and how this correlation affects prognosis still remains unclear. MethodsscRNA-seq data of ESCC were downloaded from SRA (Sequence Read Archive) database. “AUCell” algorithm was used to grouping epithelial cells according to cuproptosis and OXPHOS score. Cell-cell communication, Pseudo-time Trajectory and transcription factor enrichment analysis were repectively conducted by “CellChat”, “monocle3” package and “pySCENIC” algorithm. Univariate and LASSO cox regression analysis were used to construct the prognostic cuproptosis-OXPHOS signature. Finally, CCK-8 assay and DCFH-DA staining assay were respectively validated the sensitive and ROS production of elesclomol. ResultsscRNA-seq data were analyzed to identify 10 core cell types. According to the median scores for cuproptosis and OXPHOS, malignant epithelial cells were divided into double high, double low, and mixed groups. The double high group distributed at the end of the pseudo-time trajectory and harbored HMGA1(+) as specific transcriptional regulons. Knockdown of HMGA1 partly reversed the inhibition of cell viability visualized by CCK-8 assay, while reactive oxygen species (ROS) production by elesclomol was enhanced after HMGA1 silencing. Furthermore, the immunosuppressive signal was significantly increased in the double high group detected by ‘CellChat’ in single-cell data and ‘ssGSEA’ in bulk data followed by ‘CIBERSORTx’ algorithm. Finally, a new cuproptosis-OXPHOS prognostic signature (CNN2, ATP6V0E1, PSMD6, CCDC25, IGFBP2, MT1E, and RPS4Y1) was constructed for the prediction of the prognosis, and a high-risk group corresponding to a more sensitive tendency to erlotinib, dasatinib, and bosutinib treatment was identified. ConclusionsOur study revealed the relationship between OXPHOS and tendency of cuproptosis in ESCC, and malignant cells with this characteristic exerted immunosuppressive signals and indicated poor prognosis. Furthermore, we constructed the regulatory network in high cuproptosis-OXPHOS ESCC and identified HMGA1 as a potential regulator molecule of cuproptosis mediated by elesclomol.

Systematic identification of genotype-dependent enhancer variants in eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a rare atopic disorder associated with esophageal dysfunction, including difficulty swallowing, food impaction, and inflammation, that develops in a small subset of people with food allergies. Genome-wide association studies (GWASs) have identified 9 independent EoE risk loci reaching genome-wide significance (p<5×10-8) and 27 additional loci of suggestive significance (5×10-8<p<1×10-5). In the current study, we perform linkage disequilibrium (LD) expansion of these loci to nominate a set of 531 variants that are potentially causal. To systematically interrogate the gene regulatory activity of these variants, we designed a massively parallel reporter assay (MPRA) containing the alleles of each variant within their genomic sequence context cloned into a GFP reporter library. Analysis of reporter gene expression in TE-7, HaCaT, and Jurkat cells revealed cell-type-specific gene regulation. We identify 32 allelic enhancer variants, representing 6 genome-wide significant EoE loci and 7 suggestive EoE loci, that regulate reporter gene expression in a genotype-dependent manner in at least one cellular context. By annotating these variants with expression quantitative trait loci (eQTL) and chromatin looping data in related tissues and cell types, we identify putative target genes affected by genetic variation in individuals with EoE. Transcription factor enrichment analyses reveal possible roles for cell-type-specific regulators, including GATA3. Our approach reduces the large set of EoE-associated variants to a set of 32 with allelic regulatory activity, providing functional insights into the effects of genetic variation in this disease.

Evidence of innate training in bovine γδ T cells following subcutaneous BCG administration.

The Bacillus Calmette Guerin (BCG) vaccine has been shown to induce non-specific protection against diseases other than tuberculosis in vaccinated individuals, attributed to the induction of trained immunity. We have previously demonstrated that BCG administration induces innate immune training in mixed peripheral blood mononuclear cells and monocytes in calves. Gamma Delta (γδ) T cells are non-conventional T cells that exhibit innate and adaptive immune system features. They are in higher proportion in the peripheral blood of cattle than humans or rodents and play an essential role in bovine immune response to pathogens. In the current study, we determined if BCG administration induced innate immune training in bovine γδ T cells. A group of 16 pre-weaned Holstein calves (2-4 d age) were enrolled in the study and randomly assigned to vaccine and control groups (n=8/group). The vaccine group received two doses of 106 colony forming units (CFU) BCG Danish strain subcutaneously, separated by 2 weeks. The control group remained unvaccinated. Gamma delta T cells were purified from peripheral blood using magnetic cell sorting three weeks after receiving the 1st BCG dose. We observed functional changes in the γδ T cells from BCG-treated calves shown by increased IL-6 and TNF-α cytokine production in response to in vitro stimulation with Escherichia coli LPS and PAM3CSK4. ATAC-Seq analysis of 78,278 regions of open chromatin (peaks) revealed that γδ T cells from BCG-treated calves had an altered epigenetic status compared to cells from the control calves. Differentially accessible peaks (DAP) found near the promoters of innate immunity-related genes like Siglec14, Irf4, Ifna2, Lrrfip1, and Tnfrsf10d were 1 to 4-fold more accessible in cells from BCG-treated calves. MOTIF enrichment analysis of the sequences within DAPs, which explores transcription factor binding motifs (TFBM) upstream of regulatory elements, revealed TFBM for Eomes and IRF-5 were among the most enriched transcription factors. GO enrichment analysis of genes proximal to the DAPs showed enrichment of pathways such as regulation of IL-2 production, T-cell receptor signaling pathway, and other immune regulatory pathways. In conclusion, our study shows that subcutaneous BCG administration in pre-weaned calves can induce innate immune memory in the form of trained immunity in γδ T cells. This memory is associated with increased chromatin accessibility of innate immune response-related genes, thereby inducing a functional trained immune response evidenced by increased IL-6 and TNF-α cytokine production.

Malignant Transformation of Normal Oral Tissue to Dysplasia and Early Oral Squamous Cell Carcinoma: An In Silico Transcriptomics Approach.

Background: Oral squamous cell carcinoma (OSCC) is a prevalent and aggressive form of head and neck cancer, often diagnosed at advanced stages. Elucidating the molecular mechanisms involved in the malignant transformation from normal oral tissue to oral preinvasive lesions (OPL) and primary OSCC could facilitate early diagnosis and improve therapeutic strategies. Methods: Differentially expressed genes (DEGs) were identified from the GSE30784 dataset by comparing normal oral tissue, oral dysplasia, and primary OSCC samples. Cross-validation was performed using an independent RNA-seq dataset, GSE186775. Protein-protein interaction (PPI) network analysis, gene ontology annotation, and pathway enrichment analysis were conducted on the common DEGs. Hub genes were identified, and their prognostic significance was evaluated using survival analysis. Transcription factor (TF) enrichment analysis, cross-validation, and immunohistochemistry analyses were also performed. Results: A total of 226 proteins and 677 interactions were identified in the PPI network, with 34 hub genes, including FN1, SERPINE1, PLAUR, THBS1, and ITGA6. Pathways such as "Formation of the cornified envelope," "Keratinization," and "Developmental biology" were enriched. Overexpression of SERPINE1, PLAUR, THBS1, and ITGA6 correlated with poor prognosis, while upregulation of CALML5 and SPINK5 was associated with favorable outcomes. NFIB emerged as the most significant TF-regulating hub genes. Immunohistochemistry validated ITGA6 overexpression in primary OSCC. Cross-validation using the RNA-seq dataset supported the involvement of critical genes in the malignant transformation process. Conclusion: This study identified vital genes, pathways, and prognostic markers involved in the malignant transformation from normal oral tissue to OPL and primary OSCC, providing insights for early diagnosis and targeted therapy development. Cross-validation with an independent RNA-seq dataset and immunohistochemistry reinforced the findings, supporting the robustness of the identified molecular signatures.

The transcription factor VAX1 in VIP neurons of the suprachiasmatic nucleus impacts circadian rhythm generation, depressive-like behavior, and the reproductive axis in a sex-specific manner in mice.

The suprachiasmatic nucleus (SCN) within the hypothalamus is a key brain structure required to relay light information to the body and synchronize cell and tissue level rhythms and hormone release. Specific subpopulations of SCN neurons, defined by their peptide expression, regulate defined SCN output. Here we focus on the vasoactive intestinal peptide (VIP) expressing neurons of the SCN. SCN VIP neurons are known to regulate circadian rhythms and reproductive function. To specifically study SCN VIP neurons, we generated a novel knock out mouse line by conditionally deleting the SCN enriched transcription factor, Ventral Anterior Homeobox 1 (Vax1), in VIP neurons (Vax1Vip; Vax1fl/fl:VipCre). We found that Vax1Vip females presented with lengthened estrous cycles, reduced circulating estrogen, and increased depressive-like behavior. Further, Vax1Vip males and females presented with a shortened circadian period in locomotor activity and ex vivo SCN circadian period. On a molecular level, the shortening of the SCN period was driven, at least partially, by a direct regulatory role of VAX1 on the circadian clock genes Bmal1 and Per2. Interestingly, Vax1Vip females presented with increased expression of arginine vasopressin (Avp) in the paraventricular nucleus, which resulted in increased circulating corticosterone. SCN VIP and AVP neurons regulate the reproductive gonadotropin-releasing hormone (GnRH) and kisspeptin neurons. To determine how the reproductive neuroendocrine network was impacted in Vax1Vip mice, we assessed GnRH sensitivity to a kisspeptin challenge in vivo. We found that GnRH neurons in Vax1Vip females, but not males, had an increased sensitivity to kisspeptin, leading to increased luteinizing hormone release. Interestingly, Vax1Vip males showed a small, but significant increase in total sperm and a modest delay in pubertal onset. Both male and female Vax1Vip mice were fertile and generated litters comparable in size and frequency to controls. Together, these data identify VAX1 in SCN VIP neurons as a neurological overlap between circadian timekeeping, female reproduction, and depressive-like symptoms in mice, and provide novel insight into the role of SCN VIP neurons.

Human and mouse neutrophils share core transcriptional programs in both homeostatic and inflamed contexts

Neutrophils are frequently studied in mouse models, but the extent to which findings translate to humans remains poorly defined. In an integrative analysis of 11 mouse and 13 human datasets, we find a strong correlation of neutrophil gene expression across species. In inflammation, neutrophils display substantial transcriptional diversity but share a core inflammation program. This program includes genes encoding IL-1 family members, CD14, IL-4R, CD69, and PD-L1. Chromatin accessibility of core inflammation genes increases in blood compared to bone marrow and further in tissue. Transcription factor enrichment analysis implicates members of the NF-κB family and AP-1 complex as important drivers, and HoxB8 neutrophils with JunB knockout show a reduced expression of core inflammation genes in resting and activated cells. In independent single-cell validation data, neutrophil activation by type I or type II interferon, G-CSF, and E. coli leads to upregulation in core inflammation genes. In COVID-19 patients, higher expression of core inflammation genes in neutrophils is associated with more severe disease. In vitro treatment with GM-CSF, LPS, and type II interferon induces surface protein upregulation of core inflammation members. Together, we demonstrate transcriptional conservation in neutrophils in homeostasis and identify a core inflammation program shared across heterogeneous inflammatory conditions.

Subcytoplasmic location of translation controls protein output

The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.

Integration of single-cell transcriptomics and epigenetic analysis reveals enhancer-controlled TIMP1 as a regulator of ferroptosis in colorectal cancer.

Ferroptosis is an iron-dependent non-apoptotic programmed cell death. However, the regulatory mechanism of ferroptosis in colorectal cancer (CRC) is still unclear. The aim of this study was to investigate the role and mechanism of enhancer-controlled genes in ferroptosis in CRC. Dimensionality reduction and differentially expressed genes (DEGs) identification were conducted using Seurat algorithm based on single-cell RNA sequencing (scRNA-seq) data from the GSE200997 dataset. Ferroptosis-related pathway enrichment analysis was performed using the FerrDb V2 database. Enhancers were identified using HOMER algorithm based on H3K27ac ChIP-seq data from the GSE166254 dataset. Kaplan-Meier Plotter online tool was used to analyze prognosis and gene expression correlation. Transcription factors were predicted using the transcription factor affinity prediction web tool. The binding of enhancer to transcription factor and H3K27ac enrichment were detected by ChIP-qPCR. RSL3 was used to induce ferroptosis in CRC cells. Gene transcription was detected by qRT-PCR. Cell proliferation was detected by CCK8 assay. Nine cell clusters including T cells, natural killer cells, macrophages, mast cells, epithelial cells, fibroblasts, goblet cells, B cells and dendritic cells were identified in CRC and normal colonic tissue samples. Compared to normal colonic tissue-derived epithelial cells, 1075 DEGs were screened in CRC tissue-derived epithelial cells. Ferroptosis-related pathway enrichment suggested that DEGs were associated with the regulation of ferroptosis. DPEP1, ETV4, CEBPG, TIMP1, DUOX2 and LCN2 were identified as the significantly upregulated genes enriched in the "ferroptosis regulator" term, and their H3K27ac signals were significantly higher in CRC tissues than in normal colonic tissues. Of these, only the expression of TIMP1 predicted a poor prognosis of CRC patients. Transcription factor SPI1 drove TIMP1 transcription by binding to its enhancer. Overexpression of TIMP1 significantly promoted the resistance to ferroptosis induced by RSL3 in CRC cells, which was partially restored by SPI1 knockdown. Transcription of TIMP1 was driven by transcription factor SPI1 in combination with its enhancer, consequently promoting CRC cells against ferroptosis. The SPI1/TIMP1 axis confers ferroptosis resistance in CRC, and thus has the potential to be the molecular targets for CRC treatment.

Proteomic analysis of X-linked dystonia parkinsonism disease striatal neurons reveals altered RNA metabolism and splicing

X-linked dystonia-parkinsonism (XDP) is a rare neurodegenerative disease endemic to the Philippines. The genetic cause for XDP is an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within intron 32 of TATA-binding protein associated factor 1 (TAF1) that causes an alteration of TAF1 splicing, partial intron retention, and decreased transcription. Although TAF1 is expressed in all organs, medium spiny neurons (MSNs) within the striatum are one of the cell types most affected in XDP. To define how mutations in the TAF1 gene lead to MSN vulnerability, we carried out a proteomic analysis of human XDP patient-derived neural stem cells (NSCs) and MSNs derived from induced pluripotent stem cells. NSCs and MSNs were grown in parallel and subjected to quantitative proteomic analysis in data-independent acquisition mode on the Orbitrap Eclipse Tribrid mass spectrometer. Subsequent functional enrichment analysis demonstrated that neurodegenerative disease-related pathways, such as Huntington's disease, spinocerebellar ataxia, cellular senescence, mitochondrial function and RNA binding metabolism, were highly represented. We used weighted coexpression network analysis (WGCNA) of the NSC and MSN proteomic data set to uncover disease-driving network modules. Three of the modules significantly correlated with XDP genotype when compared to the non-affected control and were enriched for DNA helicase and nuclear chromatin assembly, mitochondrial disassembly, RNA location and mRNA processing. Consistent with aberrant mRNA processing, we found splicing and intron retention of TAF1 intron 32 in XDP MSN. We also identified TAF1 as one of the top enriched transcription factors, along with YY1, ATF2, USF1 and MYC. Notably, YY1 has been implicated in genetic forms of dystonia. Overall, our proteomic data set constitutes a valuable resource to understand mechanisms relevant to TAF1 dysregulation and to identify new therapeutic targets for XDP.

Ex Vivo Mathematical Myeloma Advisor (EMMA) - a Clinical, Molecular, and Phenotypic Platform to Tailor Personalized Therapeutic Strategies for Multiple Myeloma

Multiple myeloma (MM) is a genetically heterogeneous disease, lacking molecular markers for patient stratification and prediction of clinical response. We have created a unique platform for clinical-informed decisions, research advancement, and preclinical compound testing called the “Ex Vivo Mathematical Myeloma Advisor” (EMMA). In this pipeline, MM patients who undergo bone marrow (BM) biopsy and consent to participate in the Total Cancer Care protocol have their clinical data (demographics, therapies received, lab results, clinical history, etc.) abstracted, along with the molecular characterization (FISH/cytogenetics, RNA-Seq, and WES) of their disease. In parallel, fresh BM-derived MM blasts are combined with stromal cells in a collagen-based matrix containing patient plasma and used in an ex vivo drug screening assay. The cells are seeded into 384 or 1536-well plates and exposed to serial dilutions of up to 127 single agents and combinations. The entire setup is imaged in bright field every 30 minutes for a duration of 6 days. Image analysis is used to convert membrane integrity into survival curves, and AUC and LD50 values are estimated. The pattern of these curves facilitates the identification of resistant subclones. This data is further combined with augmented synergy models that are parameterized with PK/PD data for each drug. This integration permits the simulation of “in silico clinical trials”, which are compiled into reports that assist clinicians in selecting the best treatment for their patients. In addition, the immune population of the BM aspirate is characterized by flow cytometry, and soluble factors in the patient's BM plasma are quantified by ELISA. The integration of clinical, molecular, and phenotypic data with image analysis and machine learning tools presents a unique opportunity for finding molecular signatures of drug response (Fig. 1). For instance, our pipeline can be utilized to identify clinical factors (such as ethnicity, age, etc.) and molecular features (mutations, cytogenetic abnormalities, enriched transcription factors, differentially expressed biological pathways, etc.) associated with drug resistance/sensitivity. Additionally, it enables the determination of the mechanisms of action of new compounds and the prediction of potential synergistic/antagonistic drug combinations. The longitudinal acquisition of data in our non-destructive ex vivo drug screen assay facilitates the exploration of drug interaction dynamics over time, enabling the pinpointing of the moment when synergism occurs at maximum intensity. This, in turn, allows us to propose optimized temporal-based therapeutic regimens for clinical use. Up to this point, our pipeline has molecularly characterized nearly 1,000 MM samples, and EMMA has been applied to over 600 samples, forming one of the world's most extensive and comprehensively characterized MM cohorts. While EMMA has proven effective in quantifying the activity of monoclonal antibodies, ongoing efforts are focusing on incorporating individual immune cell components to assess ex vivo activity in the context of evolving cellular immunotherapies. As a proof of principle, we demonstrate the system's capability to measure the effect of daratumumab (CD38+ antibody) on autologous macrophage-mediated MM cell death, thereby distinguishing it from apoptosis caused by direct antibody binding (Fig. 2). Additionally, we evaluate the influence of teclistamab (T-cell engager CD3/BCMA bispecific antibody) on CD8+ T cell activity against autologous MM cells, as well as the efficacy of CAR-T cells in killing MM cells. Through the testing of standard-of-care drugs alongside immune agents, our pipeline has identified the combination of bortezomib + daratumumab as a synergistic approach with significant translational potential, warranting further investigation. In summary, our state-of-the-art platform facilitate drug discovery and unravel the intricate biological phenomena underlying drug interactions in MM. The advantages offered by our system are threefold: i) it enables pharmaceutical companies to test new compounds in a bona fide, patient-derived model; ii) it assists clinicians in selecting the best therapy for their patients, and iii) it helps prevent cancer patients from receiving non-suitable therapies. This approach can also be promptly applied to other liquid cancers, such as leukemia and lymphoma.