Combined RNA Sequencing Research Articles

INO80-Dependent Remodeling of Transcriptional Regulatory Network Underlies the Progression of Heart Failure.

Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of transcriptional network switching from normal to failed hearts remain to be determined. In this study, we integrated human samples, genetic mouse models, and genomic approaches, including bulk RNA sequencing, single-cell RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, to identify the role of chromatin remodeling complex INO80 in heart homeostasis and dysfunction. The INO80 chromatin remodeling complex was abundantly expressed in mature cardiomyocytes, and its expression further increased in mouse and human heart failure. Cardiomyocyte-specific overexpression of Ino80, its core catalytic subunit, induced heart failure within 4 days. Combining RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, we revealed INO80 overexpression-dependent reshaping of the nucleosomal landscape that remodeled a core set of transcription factors, most notably the MEF2 (Myocyte Enhancer Factor 2) family, whose target genes were closely associated with cardiac function. Conditional cardiomyocyte-specific deletion of Ino80 in an established mouse model of heart failure demonstrated remarkable preservation of cardiac function. In summary, our findings shed light on the INO80-dependent remodeling of the chromatin landscape and transcriptional networks as a major mechanism underlying cardiac dysfunction in heart failure, and suggest INO80 as a potential preventative or interventional target.

Systematic analysis of apoptosis-related genes in the prognosis of lung squamous cell carcinoma: a combined single-cell RNA sequencing study.

Lung squamous cell carcinoma (LUSC) has a poor prognosis and lacks appropriate diagnostic and treatment strategies. Apoptosis dysregulation is associated with tumor occurrence and drug resistance, but the prognostic value of apoptosis-related genes (ARGs) in LUSC remains unclear. Using univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and multivariate Cox regression analysis based on differentially expressed ARGs, we constructed an ARG-related prognostic model for LUSC survival rates. We conducted correlation analysis of prognostic ARGs by incorporating the dataset of normal lung tissue from the Genotype-Tissue Expression (GTEx) database. We then constructed a risk model, and the predictive ability of the model was evaluated using receiver operating characteristic (ROC) curve analysis. Non-small cell lung cancer (NSCLC) single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Subsequently, these data were subjected to single-cell analysis. Cell subgroups were determined and annotated by dimensionality reduction clustering, and the cell subgroups in disease development were identified via pseudotemporal analysis with the Monocle 2 algorithm. We identified four significantly prognostic ARGs and constructed a stable prognostic risk model. Kaplan-Meier curve analysis showed that the high-risk group had a poorer prognosis (P<0.05). Furthermore, the ROC analysis of 3-, 5- and 7-year survival rates confirmed that the model had good predictive value for patients with LUSC. Single-cell RNA sequencing showed the prognostic ARGS were enriched in epithelial cells, smooth muscle cells, and T cells. Pseudotime analysis was used to infer the differentiation process and time sequence of cells. This study identified ARGs that are associated with prognosis in LUSC, and a risk model based on these prognostic genes was constructed that could accurately predict the prognosis of LUSC. Single-cell sequencing analysis provided new insights into the cellular-level development of tumors. These findings provide more guidance for the diagnosis and treatment of patients with LUSC.

Hyperglycolysis in endothelial cells drives endothelial injury and microvascular alterations in peritoneal dialysis.

Endothelial cell (EC) dysfunction leading to microvascular alterations is a hallmark of technique failure in peritoneal dialysis (PD). However, the mechanisms underlying EC dysfunction in PD are poorly defined. We combined RNA sequencing with metabolite set analysis to characterize the metabolic profile of peritoneal ECs from a mouse model of PD. This was combined with EC-selective blockade of glycolysis by genetic or pharmacological inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in vivo and in vitro. We also investigated the association between peritoneal EC glycolysis and microvascular alterations in human peritoneal samples from patients with end-stage kidney disease (ESKD). In a mouse model of PD, peritoneal ECs had a hyperglycolytic metabolism that shunts intermediates into nucleotide synthesis. Hyperglycolytic mouse peritoneal ECs displayed a unique active phenotype with increased proliferation, permeability and inflammation. The active phenotype of mouse peritoneal ECs can be recapitulated in human umbilical venous ECs and primary human peritoneal ECs by vascular endothelial growth factor that was released from high glucose-treated mesothelial cells. Importantly, reduction of peritoneal EC glycolysis, via endothelial deficiency of the glycolytic activator PFKFB3, inhibited PD fluid-induced increases in peritoneal capillary density, vascular permeability and monocyte extravasation, thereby protecting the peritoneum from the development of structural and functional damages. Mechanistically, endothelial PFKFB3 deficiency induced the protective effects in part by inhibiting cell proliferation, VE-cadherin endocytosis and monocyte-adhesion molecule expression. Pharmacological PFKFB3 blockade induced a similar therapeutic benefit in this PD model. Human peritoneal tissue from patients with ESKD also demonstrated evidence of increased EC PFKFB3 expression associated with microvascular alterations and peritoneal dysfunction. These findings reveal a critical role of glycolysis in ECs in mediating the deterioration of peritoneal function and suggest that strategies targeting glycolysis in peritoneal ECs may be of therapeutic benefit for patients undergoing PD.

Instructive Interaction between Myelodysplastic Hematopoiesis and the Bone Marrow Microenvironment at Single Cell Level

Myelodysplastic Neoplasms (MDS) are hypothesized to re-model their bone marrow (BM) microenvironment to re-inforce conditions for their propagation. In this study, we investigated interactions between MDS cells and the BM niche at single cell level in a MDS patient-derived xenograft model (PDX) as well as primary human MDS and healthy (HY) bone marrow (BM samples in order to identify cues of disturbed crosstalk between MDS hematopoiesis and the BM microenvironment. In the PDX model, we analyzed &amp;gt;13,000 cells of n=15 different murine niche cell populations after long-term (&amp;gt;24 weeks) exposure to n=3 MDS patient samples versus n=2 age-matched HY human grafts. Subsequently, we analyzed over 24,000 primary human BM cells enriched for the non-hematopoietic compartment by using whole bone fragments from n=8 MDS patients and n=7 HY age-matched donors. All cells were analyzed with combined 10x single cell RNA sequencing and human “Cellular Indexing of the Transcriptome and Epitopes by Sequencing” (CITE Seq). Results were validated by flow cytometry and gene expression analyses. Mesenchymal cell (MSC) subpopulations from PDX mice exposed to MDS versus HY hematopoiesis could be subdivided into distinct “Cxcl12+ abundant reticular” (CAR) cells with osteogenic and adipogenic differentiation trajectories. Various CAR cell populations displayed an increased frequency in MDS transplanted mice as compared to PDX transplanted with HY grafts ( Panel A). Upon contact with hematopoietic MDS cells, CAR cell populations overexpressed numerous genes involved in extracellular matrix reorganization and hematopoietic supporting factors, such as Cxcl12, Il7, Lox and Loxl1 as compared to mice bearing HY grafts. In situ, BM cells from MDS patients showed highly heterogeneous MSC subpopulations with similar differentiation trajectories to the ones observed in our PDX models on a patient-individual level. We detected two non-hematopoietic cell populations, designated as endothelial cells (ECs) and MSCs characterized by expression of CXCL12, IGFBP5 or COL1A2 and quantification with epitope sequencing (CITE Seq) with mutually exclusive detection of CD31 and CD271 expression (n=581 cells in total, Panel B). In n=3 MDS patients, we identified an MDS-specific MSC subpopulation with high IGFPB4 and CXCL12 expression as well as overexpression of KITLG and IL7. This suggests that this altered specialized secretory mesenchymal state is present in a subset of MDS patients and may support the malignant hematopoietic clones by overexpression of key hematopoietic factors. Interactome analyses using spatial receptor:ligand bioinformatic tools such as CellphoneDB, RNAMagnet and omnipath further confirmed that stromal cells alter their cellular profile and secretory behavior in response to MDS hematopoiesis. In conclusion, we show that murine BM stroma is re-programmed by exposure to human MDS xenografts to upregulate extracellular matrix (ECM) re-modeling and HSC supporting factors. In native human MDS BM in vivo, rare primary mesenchymal cells are re-programmed to increase hematopoietic support in a patient-individual manner pointing to altered MSC subpopulations with increased growth factor expression profiles in a subgroup of MDS patients.

Acetyl-CoA is a key molecule for nephron progenitor cell pool maintenance

Nephron endowment at birth impacts long-term renal and cardiovascular health, and it is contingent on the nephron progenitor cell (NPC) pool. Glycolysis modulation is essential for determining NPC fate, but the underlying mechanism is unclear. Combining RNA sequencing and quantitative proteomics we identify 267 genes commonly targeted by Wnt activation or glycolysis inhibition in NPCs. Several of the impacted pathways converge at Acetyl-CoA, a co-product of glucose metabolism. Notably, glycolysis inhibition downregulates key genes of the Mevalonate/cholesterol pathway and stimulates NPC differentiation. Sodium acetate supplementation rescues glycolysis inhibition effects and favors NPC maintenance without hindering nephrogenesis. Six2Cre-mediated removal of ATP-citrate lyase (Acly), an enzyme that converts citrate to acetyl-CoA, leads to NPC pool depletion, glomeruli count reduction, and increases Wnt4 expression at birth. Sodium acetate supplementation counters the effects of Acly deletion on cap-mesenchyme. Our findings show a pivotal role of acetyl-CoA metabolism in kidney development and uncover new avenues for manipulating nephrogenesis and preventing adult kidney disease.

Single-Cell Transcriptomics Points to a Role of Stemness and Bone Marrow Niche Interactions in the Poor Response to Induction Therapy in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis with a 15-20% relapse rate. The induction therapy response is the strongest predictor of relapse and shows great variation between patients. However, whether intra-patient leukemic cell heterogeneity plays a role for the induction therapy response has not yet been investigated. We studied 13 patients with T-ALL (11 pediatric and 2 young adult, median follow-up 37 months). Of these, three were good responders (end of induction (EOI) minimal residual disease (MRD) undetectable/non-quantifiable by flowcytometry and PCR), while ten had a poor response (EOI MRD&amp;gt;0.01%, (range 0.05-97%). Of the ten poor responders, three experienced relapse and one died from resistant disease. Bone marrow samples from diagnosis (n=13) and paired samples from relapse (n=3) or MRD (n=4, MRD &amp;gt;5%) were subjected to combined single-cell RNA sequencing, single-cell surface marker profiling by antibody-derived tag (ADT)-sequencing, and single-cell TCR sequencing, using the 10X Genomics platform. A published healthy bone marrow dataset was used for cell annotation and clear distinction was observed between healthy cell subsets and leukemic blasts. TCR sequencing data revealed a hyperexpanded clone in six patients, with identical clonal sequences at diagnosis and relapse(n=2)/MRD(n=1). Surface marker profiling using a panel of 134 surface antigens showed good correspondence between leukemia-associated immunophenotypes found by ADT-sequencing and by flow cytometry for 16 common clinically used markers. To examine factors associated with a poor therapy response, we compared average gene expression of leukemic cells in poor and good responders at diagnosis using differential gene expression (DGE) and gene set enrichment analysis (GSEA). At diagnosis, poor responders showed upregulation of genes involved in extracellular matrix (ECM) degradation/organization, including the adhesion molecules ITGA5 and PECAM1, and MMP11, while showing downregulation of cell cycle genes. Accordingly, the fraction of cells predicted to be in G0/G1 phase at diagnosis of poor responders positively correlated with EOI MRD (r=0.52, p=0.07, Spearman's rank correlation). To investigate if this high fraction of quiescent cells in poor responders was associated with a more stem-like cell state, we examined the expression of a published hematopoietic stem cell (HSC) signature (Forsberg et al, 2010) that we previously selected for investigation in BCP-ALL (Modvig et al, Molecular Oncology, 2022). Indeed, patients with a poor response had a higher percentage of leukemic blasts with an HSC signature module score above zero (p=0.028, Kolmogorov-Smirnov, distribution in Figure 1), suggestive of stemness as a contributing factor to therapy resistance. To define molecular changes in leukemic blasts during therapy, we compared the leukemic blast gene expression profiles in matched diagnostic and MRD samples using DGE and GSEA. This revealed that the leukemic blasts within the first month of therapy downregulated cell cycle genes, while upregulating chemokine receptors, such as CXCR4. To identify specific genes associated with high MRD, we combined genes upregulated in poor responders (n=314, p&amp;lt;0.01, DGE) with genes associated with EOI MRD levels (n=98, r&amp;gt;0.8 and p&amp;lt;0.001, rank regression). This resulted in a 30-gene signature, comprising several genes previously associated with cancer stem cells, such as MICAL3, ELF4, and NLRP3, and genes encoding proteins involved in cancer drug resistance. These included sialyl transferases, acid ceramidase ( ASAH1) and zyxin ( ZYX). ZYX silencing was previously shown to downregulate the anti-apoptotic protein BCL-2 in leukemia. Accordingly, the percentage of cells with detectable BCL2 expression at diagnosis correlated with EOI MRD (r=0.58, p=0.04), supporting a role of BCL-2 inhibitor therapy in T-ALL. In conclusion, our data suggest that therapy-resistant cells are more stem-like and quiescent at diagnosis, display upregulation of genes involved in bone marrow niche ECM organization and adhesion, and genes related to drug resistance. Likewise, residual leukemic blast following therapy show less proliferation and CXCR4 upregulation. Thus, single-cell transcriptomics provides important molecular insights into therapy-resistant leukemic cells and could comprise a valuable prognostic tool in T-ALL.

Distinct Patterns of CD4+ and CD8+ T-Cell Clonal Expansion Enable Broad Clinical Responses to Pembrolizumab + GVD in Patients with Relapsed Hodgkin Lymphoma

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by immune dysregulation, with recurrent amplifications of the 9p24 locus encoding several genes including CD274 (PD-L1), PDCD1LG2 (PD-L2), and JAK2, frequent genetic and epigenetic downregulation of major histocompatibility complex (MHC)-I and (MHC)-II, and a tumor microenvironment (TME) defined by T-cell infiltration and high rates of PD-L1 expression. PD-1 blockade is highly active both as single agent and in combination with chemotherapeutic agents in relapsed/refractory (RR) cHL. However, the mechanism by which PD-1 blockade promotes clinical responses in combination with chemotherapy, and whether the mechanism of action varies based on tumor or TME-specific expression of either MHC or PD-L1 remains unclear. Methods: We previously conducted a phase II study in patients with relapsed or refractory cHL evaluating pembrolizumab in combination with gemcitabine, vinorelbine and liposomal doxorubicin (P-GVD) before proceeding to high-dose therapy and autologous hematopoietic cell transplantation (AHCT) (Moskowitz AJ et al, JCO 2021). Treatment consisted of 2 to 4 cycles of P-GVD followed by AHCT. We performed combined single-cell surface protein identification, RNA sequencing, and (T cell receptor) TCR sequencing using the 10x Genomics Single Cell Immune Profiling Platform on paired C1D1 and C3D1 peripheral blood mononuclear cells (PBMCs) from 23 patients who received P-GVD and 5 control patients with relapsed cHL treated with standard chemotherapy regimens. Expression of MHC-I, MHC-II, and PD-L1 on both Hodgkin Reed Sternberg (HRS) cells and within the TME was assessed by immunohistochemistry. Results:Of 38 evaluable pts, the complete response (CR) rate after 2 or 4 cycles of P-GVD was 95%. After a median follow-up of 40 months (range: 2-56), only 1 pt experienced relapse 23 months after AHCT and another pt died 41 months after AHCT while in remission due to unrelated causes. The estimated 40-month progression-free survival (PFS) is 96% (95% CI: 91-100). CITE-Sequencing of PBMCs across 56 samples from 28 patients revealed predominantly CD4+ T, CD8+ T, NK, and myeloid cells within the peripheral blood, with smaller proportions of hematopoietic progenitors and plasmacytoid dendritic cells. Combined RNA and TCR analysis of the T-cell compartment revealed significant pre-treatment clonal expansion of both CD4+ and CD8+ T-cells exhibiting a cytotoxic phenotype, including expression of GZMH, KLRG1, and CCL5 by CD8+ T-cells and expression of PRF1, NKG7, GNLY, FGFBP2, and GZMH by CD4+ T-cells ( Fig. 1A). Correlation of pre-treatment clonal expansion with tumor-specific MHC status demonstrated that clonal expansion of CD8+ T-cells was more robust in patients whose tumors expressed either MHC-I or MHC-II; conversely, clonal expansion of CD4+ T-cells was most robust in tumors expressing MHC-II but lacking MHC-I, suggesting cytotoxic CD4+ T-cell responses may promote anti-tumor immunity in MHC-I-deficient tumors ( Fig. 1B). Both CD4+ and CD8+ T-cell clonal expansion was highest in patients whose tumors expressed high levels of PD-L1 on stromal cells. Treatment with P-GVD did not result in further clonal expansion of either CD4+ or CD8+ T-cells but did increase expression of cytotoxic genes in pre-treatment expanded CD4+ T-cells, including NKG7 and PLEK, while downregulating negative genes of T-cell function in pre-treatment expanded CD8+ T-cells, including TNFAIP3 and DUSP1, suggesting that P-GVD promotes anti-tumor immunity in cHL by enhancing the cytotoxicity of tumor-reactive expanded CD4+ and CD8+ T-cells. Conclusions: Treatment of relapsed cHL with P- GVD resulted in durable remissions in patients undergoing AHCT. Distinct patterns of peripheral blood T-cell expansion were observed based on MHC-I and MHC-II expression on tumor cells, with CD4+ T-cell clonal expansion uniquely expanding in patients with MHC-I-deficient tumors. Further analysis of the impact of P-GVD on T-cell differentiation and function is ongoing. AcknowledgementT: Supported in part by a research grant from Investigator-Initiated Studies Program of Merck Sharp &amp; Dohme LLC. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp &amp; Dohme LLC.

Transcriptional profiling of dental sensory and proprioceptive trigeminal neurons using single-cell RNA sequencing

Dental primary afferent (DPA) neurons and proprioceptive mesencephalic trigeminal nucleus (MTN) neurons, located in the trigeminal ganglion and the brainstem, respectively, are essential for controlling masticatory functions. Despite extensive transcriptomic studies on various somatosensory neurons, there is still a lack of knowledge about the molecular identities of these populations due to technical challenges in their circuit-validated isolation. Here, we employed high-depth single-cell RNA sequencing (scRNA-seq) in combination with retrograde tracing in mice to identify intrinsic transcriptional features of DPA and MTN neurons. Our transcriptome analysis revealed five major types of DPA neurons with cell type-specific gene enrichment, some of which exhibit unique mechano-nociceptive properties capable of transmitting nociception in response to innocuous mechanical stimuli in the teeth. Furthermore, we discovered cellular heterogeneity within MTN neurons that potentially contribute to their responsiveness to mechanical stretch in the masseter muscle spindles. Additionally, DPA and MTN neurons represented sensory compartments with distinct molecular profiles characterized by various ion channels, receptors, neuropeptides, and mechanoreceptors. Together, our study provides new biological insights regarding the highly specialized mechanosensory functions of DPA and MTN neurons in pain and proprioception.

Genome-wide screening reveals metabolic regulation of stop-codon readthrough by cyclic AMP.

Translational fidelity is critical for microbial fitness, survivaland stress responses. Much remains unknown about the genetic and environmental control of translational fidelity and its single-cell heterogeneity. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified over 20 genes critical for stop-codon readthrough. Most of these identified genes were not previously known to affect translational fidelity. Intriguingly, we show that several genes controlling metabolism, including cyaA and crp, enhance stop-codon readthrough. CyaA catalyzes the synthesis of cyclic adenosine monophosphate (cAMP). Combining RNA sequencing, metabolomicsand biochemical analyses, we show that deleting cyaA impairs amino acid catabolism and production of ATP, thus repressing the transcription of rRNAs and tRNAs to decrease readthrough. Single-cell analyses further show that cAMP is a major driver of heterogeneity in stop-codon readthrough and rRNA expression. Our results highlight that carbon metabolism is tightly coupled with stop-codon readthrough.

Intratumoral CD103+ CD8+ T cells predict response to neoadjuvant chemoimmunotherapy in advanced head and neck squamous cell carcinoma.

Immune cell heterogenicity is known to determine the therapeutic response to cancer progression. Neoadjuvant chemoimmunotherapy (NACI) has shown clinical benefits in some patients with advanced head and neck squamous cell carcinoma (HNSCC), but the underlying mechanism behind this clinical response is unknown. The efficacy of NACI needs to be potentiated by identifying accurate biomarkers to predict clinical responses. Here, we attempted to identify molecules predicting NACI response in advanced HNSCC. We performed combined single-cell RNA sequencing (scRNA-seq) and multiplex immunofluorescence (mIHC) staining with tumor samples derived from NACI-treated HNSCC patients to identify a new tumor-infiltrating cell (TIL) subtype, CD103+ CD8+ TILs, associated with clinical response, while both in vitro and in vivo assays were carried out to determine its antitumor efficiency. The regulatory mechanism of the CD103+ CD8+ TILs population was examined by performing cell-cell interaction analysis of the scRNA-seq data and spatial analysis of the mIHC images. We established intratumoral CD103+ CD8+ TILs density as a determinant of NACI efficacy in cancers. Our scRNA-seq results indicated that the population of CD103+ CD8+ TILs was dramatically increased in the responders of NACI-treated HNSCC patients, while mIHC analysis confirmed the correlation between intratumoral CD103+ CD8+ TILs density and NACI efficacy in HNSCC patients. Further receiver operating characteristic curve analysis defined this TIL subset as a potent marker to predict patient response to NACI. Functional assays showed that CD103+ CD8+ TILs were tumor-reactive T cells, while programmed cell death protein-1 (PD-1) blockade enhanced CD103+ CD8+ TILs cytotoxicity against tumor growth in vivo. Mechanistically, targeting the triggering receptor expressed on myeloid cells 2-positive (TREM2+ ) macrophages might enhance the population of CD103+ CD8+ TILs and facilitate antitumor immunity during NACI treatment. Our study highlights the impact of intratumoral CD103+ CD8+ TILs density on NACI efficacy in different cancers, while the efforts to elevate its population warrant further clinical investigation.

Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius.

Heat shock response is the ability to respond adequately to sudden temperature increases that could be harmful for cellular survival and fitness. It is crucial for microorganisms living in volcanic hot springs that are characterized by high temperatures and large temperature fluctuations. In this study, we investigated how S. acidocaldarius, which grows optimally at 75°C, responds to heat shock by altering its gene expression and protein production processes. We shed light on which cellular processes are affected by heat shock and propose a hypothesis on underlying regulatory mechanisms. This work is not only relevant for the organism's lifestyle, but also with regard to its evolutionary status. Indeed, S. acidocaldarius belongs to the archaea, an ancient group of microbes that is more closely related to eukaryotes than to bacteria. Our study thus also contributes to a better understanding of the early evolution of heat shock response.

Liver metastases across cancer types sharing tumor environment immunotolerance can impede immune response therapy and immune monitoring

BackgroundHepatic immune tolerance might contribute to the development of therapeutic resistance to immunotherapy. However, addressing this issue is challenging since the efficacy of immunotherapy in the context of liver metastasis (LM) remains poorly studied. Here, we aimed to establish an LM common immune feature (LMCIF) score to quantify the characteristics of LM immunotolerance across cancer types for assisting clinical disease management. MethodsLarge-scale clinical data were collected to identify the prognosis of LM. Multi-omics datasets of metastatic cancers with LM special immune-related pathways (LMSIPs) from the Molecular Signatures Database (MSigDB)were used to obtain an LMCIF cluster. Based on differential expression genes (DEGs), a novel LMCIF score for the LMCIF cluster was constructed. In addition, multi-omics, and immunohistochemistry (IHC) data from the public and in-house cohorts were used to explore the features of LM, and LMCIF score. ResultsPatients with LM had a worse prognosis and significantly lower infiltration of immune cells than patients with metastasis to other organs when analyzed with combined clinical and RNA sequencing data. After extracting the LMCIF cluster from 373 samples by utilizing 29 LMSIPs and validating them in a microarray cohort, an LMCIF score was established to confirm the role of the immunosuppressive environment as a contributor to the poor prognosis of LM across cancer types. Moreover, this LMCIF score could be used to predict the immune response of cancer patients undergoing immunotherapy. Finally, we identified that the majority of the 31 LMCIF genes exhibited a negative correlation with TME cells in LM patients, one of them, KRT19, which possessed the strongest positive correlation with LMCIF score, was confirmed to have an immunosuppressive effect through IHC analysis. ConclusionsOur results suggest that LM across cancer types share similar immunological profiles that induce immunotolerance and escape from immune monitoring. The novel LMCIF score represents a common liver metastasis immune cluster for predicting immunotherapy response, the results of which might benefit clinical disease management.

Traditional Chinese Medicine Shi-Bi-Man regulates lactic acid metabolism and drives hair follicle stem cell activation to promote hair regeneration

BackgroundAs a supplement for promoting hair health, Shi-Bi-Man (SBM) is a prescription comprising various traditional Chinese medicines. Though SBM has been reported to promote hair regeneration, its molecular mechanism remains unclear. Cynomolgus monkeys (Macaca fascicularis) are non-human primates with a gene expression profile similar to that of humans. The purpose of this research is to evaluate the effect of SBM on promoting hair regeneration in cynomolgus monkeys and to reveal the underlying mechanism.MethodsThe effect of SBM on hair regeneration was observed by skin administration on 6 cynomolgus monkeys with artificial back shaving. The molecular mechanism of SBM was studied using single-cell RNA sequencing (scRNA-seq) in combination with quantitative polymerase chain reaction (qPCR) detection for gene transcription level, and immunofluorescence staining verification for protein level.ResultsSBM significantly induced hair regeneration in cynomolgus monkeys, increased hair follicle number and facilitated hair follicle development. ScRNA-seq revealed an increase in the number of hair follicle stem cells (HFSCs) with a higher activation state, as evidenced by the higher expression of activation marker LDHA related to metabolism and the proliferation marker MKI67. Immunofluorescence analysis at the protein level and qPCR at the mRNA level confirmed the sequencing data. Cellchat analysis revealed an enrichment of ligand-receptor pairs involved in intercellular communication in Laminin-related pathways.ConclusionSBM significantly promotes hair regeneration in cynomolgus monkeys. Mechanically, SBM can up-regulate LDHA-mediated lactic acid metabolism and drive HFSC activation, which in turn promotes the proliferation and differentiation of HFSCs.

WTAP activates MAPK signaling through m6A methylation in VEGFA mRNA-mediated by YTHDC1 to promote colorectal cancer development.

N6-methyladenosine modification, especially Wilms tumor 1-associated protein (WTAP), is reportedly associated with a variety of cancers, including colorectal cancer (CRC). Angiogenesis also plays an important role in the occurrence and development of CRC. However, only a few studies have reported the biological mechanisms underlying this connection. Therefore, tissue microarray and public database were used to explore WTAP levels in CRC. Then, WTAP was down-regulated and over-expressed, respectively. CCK8, EdU, colony formation, and transwell experiments were performed to study the role of WTAP in CRC. Combined RNA sequencing and m6A RNA immunoprecipitation (MeRIP) sequencing, we found downstream molecules VEGFA. Moreover, a tube formation assay was executed for tumor angiogenesis. Finally, a subcutaneous tumorigenesis assay in nude mice was used to examine the tumor-promoting effect of WTAP in vivo. In the present study, WTAP was significantly upregulated in CRC cells and patients with CRC. Moreover, higher WTAP expression was observed in the TCGA and CPATC databases in CRC tissues. WTAP over-expression exacerbates cell proliferation, migration, invasion, and angiogenesis. Conversely, WTAP knockdown inhibited the malignant biological behavior of CRC cells. Mechanistically, WTAP positively regulated VEGFA, as identified using RNA sequencing and MeRIP sequencing. Moreover, we identified YTHDC1 as a downstream effector of the YTHDC1-VEGFA axis in CRC. Furthermore, increased WTAP expression activated the MAPK signaling pathway, which led to enhanced angiogenesis. In conclusion, our study revealed that the WTAP/YTHDC1/VEGFA axis promotes CRC development, especially angiogenesis, suggesting that it may act as a potential biomarker of CRC.

Activation of Platelet mTORC2/Akt Pathway by Anti-β2GP1 Antibody Promotes Thrombosis in Antiphospholipid Syndrome.

Anti-β2GP1 (β2-glycoprotein 1) antibodies are the primary pathogenic antibody to promote thrombosis in antiphospholipid syndrome (APS), yet the underlying mechanism remains obscure. We aimed to explore the intracellular pathway that mediated platelet activation. Platelets were isolated from patients with APS and subjected to RNA sequencing. Platelet aggregation, the release of platelet granules, platelet spreading, and clot retraction were detected to evaluate platelet activation. We purified anti-β2GP1 antibodies from patients with APS and the total IgG from healthy donors to stimulate platelets with/without FcγRIIA (Fcγ receptor IIA) blocking antibody or Akt (protein kinase B) inhibitor. Platelet-specific Sin1 (stress-activated protein kinase-interacting protein) deficiency mice were established. The thrombus model of inferior vena cava flow restriction, ferric chloride-induced carotid injury model, and laser-induced vessel wall injury in cremaster arterioles model were constructed after administration of anti-β2GP1 antibodies. Combined RNA sequencing and bioinformatics analysis suggested that APS platelets exhibited increased levels of mRNA associated with platelet activation, which was in line with the hyperactivation of APS platelets in response to stimuli. Platelet activation in APS platelets was accompanied by upregulation of the mTORC2 (mammalian target of the rapamycin complex 2)/Akt pathway and increased levels of SIN1 phosphorylation at threonine 86. Anti-β2GP1 antibody derived from patients with APS enhanced platelet activation and upregulated the mTORC2/Akt pathway. Moreover, the Akt inhibitor weakened the potentiating effect of the anti-β2GP1 antibody on platelet activation. Notably, Sin1 deficiency suppresses anti-β2GP1 antibody-enhanced platelet activation in vitro and thrombosis in all 3 models. This study elucidated the novel mechanism involving the mTORC2/Akt pathway, which mediates the promotion of platelet activation and induction of thrombosis by the anti-β2GP1 antibody. The findings suggest that SIN1 may be a promising therapeutic target for the treatment of APS.

Sargentodoxa cuneata and Patrinia villosa extract inhibits LPS-induced inflammation by shifting macrophages polarization through FAK/PI3K/Akt pathway regulation and glucose metabolism reprogramming

Ethnopharmacological relevanceSargentodoxa cuneata and Patrinia villosa (S&P) are two natural herbal medicine widely used for treatment of various inflammatory diseases in Traditional Chinese Medicine, whereas the mode of action needs to be further investigated. Aim of the studyThis study aimed to explore the anti-inflammatory effects and unravel the involved mechanism of S&P extract. Materials and methodsThe components of S&P extract were first detected using the liquid chromatography-tandem mass spectrometry (LC-MS/MS). The effects of S&P extract on the viability and migration ability of macrophages were detected using CCK8, LDH, adhesion and transwell assays. Cytokine release and macrophage phenotype transition were measured using a cytometric bead array and flow cytometry. The potential mechanism was uncovered using an integrative approach combining RNA sequencing and LC-MS/MS-based metabolic analysis. The expression of related proteins was further validated using western blotting. ResultsS&P extract inhibited the proliferation and migration of LPS-induced macrophages, changed the morphology of macrophages, and inhibited the production of NO and the expression of iNOS. Furthermore, the extract inhibited tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production and the expression of the M1 phenotype markers CD11c and CD16/32, whereas it promoted interleukin-10 (IL-10) production and the expression of the M2 phenotype markers CD206 and arginase 1 (Arg1). RNA sequencing analysis demonstrated that the upregulated genes by S&P extract treatment were involved in M2 macrophages: Il10, Ccl17, Ccl22, Cd68. The downregulated genes were involved in M1 macrophages and glycolysis processes: Stat1, Il18, Cd80, Cd86, Nos2, Il6, Pik3ap1, Raf1, Pdhb, etc. Metabolomics results showed that the S&P extract strongly ameliorated lipopolysaccharide (LPS)-induced metabolic disturbances. KEGG analysis indicated that most of these metabolites were involved in glucose metabolism, which is involved in the tumor necrosis factor (TNF), phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt), Glycolysis, and mitogen-activated protein kinase (MAPK) pathways. In vitro experiments further confirmed that the extract significantly inhibited the phosphorylation of focal adhesion kinase (FAK), PI3K and Akt, and the expression of glucose metabolism-related proteins. Adding a FAK inhibitor (defactinib) further inhibited the expression of M1/M2 phenotypic markers and the phosphorylation of FAK, PI3K, and Akt. ConclusionsS&P extract can induce M2 polarization and shift macrophages from M1 to M2 tissue repair in LPS-induced inflammation by regulating glucose metabolism and the FAK/PI3K/Akt pathway.

Transcriptional signatures of heroin intake and relapse throughout the brain reward circuitry in male mice.

Opioid use disorder (OUD) looms as one of the most severe medical crises facing society. More effective therapeutics will require a deeper understanding of molecular changes supporting drug-taking and relapse. Here, we develop a brain reward circuit-wide atlas of opioid-induced transcriptional regulation by combining RNA sequencing (RNA-seq) and heroin self-administration in male mice modeling multiple OUD-relevant conditions: acute heroin exposure, chronic heroin intake, context-induced drug-seeking following abstinence, and relapse. Bioinformatics analysis of this rich dataset identified numerous patterns of transcriptional regulation, with both region-specific and pan-circuit biological domains affected by heroin. Integration of RNA-seq data with OUD-relevant behavioral outcomes uncovered region-specific molecular changes and biological processes that predispose to OUD vulnerability. Comparisons with human OUD RNA-seq and genome-wide association study data revealed convergent molecular abnormalities and gene candidates with high therapeutic potential. These studies outline molecular reprogramming underlying OUD and provide a foundational resource for future investigations into mechanisms and treatment strategies.

Plant extracellular self-DNA inhibits growth and induces immunity via the jasmonate signaling pathway.

Plants have evolved sophisticated mechanisms to detect various forms of danger. Damage-associated molecular patterns (DAMPs) are endogenous danger molecules that are released from damaged cells and activate the innate immunity. Recent evidence suggests that plant extracellular self-DNA (esDNA) can serve as a DAMP molecule. However, the mechanisms by which esDNA functions are largely unknown. In this study, we confirmed that esDNA inhibits root growth and triggers reactive oxygen species (ROS) production in a concentration- and species-specific manner in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum L.). Furthermore, by combining RNA sequencing, hormone measurement, and genetic analysis, we found that esDNA-mediated growth inhibition and ROS production are achieved through the jasmonic acid (JA) signaling pathway. Specifically, esDNA induces JA production and the expression of JA-responsive genes. The esDNA-mediated growth inhibition, ROS production, and gene expression are impaired in the JA-related mutants. Finally, we found that the JA signaling pathway is required for the esDNA-elicited resistance against the pathogens Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000. This finding highlights the importance of JA signaling in esDNA-mediated biological effects, thereby providing insight into how esDNA functions as a DAMP.

MAIT cell inhibition promotes liver fibrosis regression via macrophage phenotype reprogramming

Recent data have shown that liver fibrosis can regress even at later stages of cirrhosis and shifting the immune response from pro-inflammatory towards a resolutive profile is considered as a promising option. The immune regulatory networks that govern the shift of the inflammatory phenotype and thus potential reversal of liver fibrosis are lesser known. Here we show that in precision-cut human liver slices obtained from patients with end-stage fibrosis and in mouse models, inhibiting Mucosal-Associated Invariant T (MAIT) cells using pharmacological or antibody-driven approaches, limits fibrosis progression and even regresses fibrosis, following chronic toxic- or non-alcoholic steatohepatitis (NASH)-induced liver injury. Mechanistic studies, combining RNA sequencing, in vivo functional studies (performed in male mice) and co-culture experiments indicate that disruption of the MAIT cell-monocyte/macrophage interaction results in resolution of fibrosis both by increasing the frequency of restorative Ly6Clo at the expenses of pro-fibrogenic Ly6Chi monocyte-derived macrophages and promoting an autophagic phenotype in both subsets. Thus, our data show that MAIT cell activation and the consequential phenotype shift of liver macrophages are important pathogenic features of liver fibrosis and could be targeted by anti-fibrogenic therapy.

Genetic diagnostic yields of 354 Chinese ASD children with rare mutations by a pipeline of genomic tests.

Purpose: To establish an effective genomic diagnosis pipeline for children with autism spectrum disorder (ASD) for its genetic etiology and intervention. Methods: A cohort of 354 autism spectrum disorder patients were obtained from Beijing Children's Hospital, Capital Medical University. Peripheral blood samples of the patients were collected for whole genome sequencing (WGS) and RNA sequencing (RNAseq). Sequencing data analyses were performed for mining the single nucleotide variation (SNV), copy number variation (CNV) and structural variation (SV). Sanger sequencing and quantitative PCR were used to verify the positive results. Results: Among 354 patients, 9 cases with pathogenic/likely pathogenic copy number variation and 10 cases with pathogenic/likely pathogenic single nucleotide variations were detected, with a total positive rate of 5.3%. Among these 9 copy number variation cases, 5 were de novo and 4 were inherited. Among the 10 de novo single nucleotide variations, 7 were previously unreported. The pathological de novo mutations account for 4.2% in our cohort. Conclusion: Rare mutations of copy number variations and single nucleotide variations account for a relatively small proportion of autism spectrum disorder children, which can be easily detected by a genomic testing pipeline of combined whole genome sequencing and RNA sequencing. This is important for early etiological diagnosis and precise management of autism spectrum disorder with rare mutations.