Interleukin-33-activated neuropeptide CGRP-producing memory Th2 cells cooperate with somatosensory neurons to induce conjunctival itch.

Allergic conjunctivitis is a chronic inflammatory disease that is characterized by severe itch in the conjunctiva, but how neuro-immune interactions shape the pathogenesis of severe itch remains unclear. We identified a subset of memory-type pathogenic Th2 cells that preferentially expressed Il1rl1-encoding ST2 and Calca-encoding calcitonin-gene-related peptide (CGRP) in the inflammatory conjunctiva using a single-cell analysis. The IL-33-ST2 axis in memory Th2 cells controlled the axonal elongation of the peripheral sensory C-fiber and the induction of severe itch. Pharmacological blockade and genetic deletion of CGRP signaling in vivo attenuated scratching behavior. The analysis of giant papillae from patients with severe allergic conjunctivitis revealed ectopic lymphoid structure formation with the accumulation of IL-33-producing epithelial cells and CGRP-producing pathogenic CD4+ T cells accompanied by peripheral nerve elongation. Thus, the IL-33-ST2-CGRP axis directs severe itch with neuro-reconstruction in the inflammatory conjunctiva and is a potential therapeutic target for severe itch in allergic conjunctivitis.

APRIL/BAFF upregulation is associated with clonal B-cell expansion in Hunner-type interstitial cystitis.

Hunner-type interstitial cystitis (HIC) is a chronic inflammatory disease of the urinary bladder with an unknown etiology. We conducted comprehensive immunogenomic profiling of bladder specimens obtained by biopsy and cystectomy from 37 patients with HIC. Next-generation RNA sequencing demonstrated abundant plasma cell infiltration with frequent light chain restriction in HIC-affected bladder tissue. Subsequent analysis of the B-cell receptor repertoire revealed spatial and temporal expansion of B-cell clones. The extent of B-cell clonal expansion was significantly correlated with the gene expression levels of TNFSF13 and TNFSF13B, which encode APRIL and BAFF, respectively. These findings indicate that APRIL and BAFF are the key regulators of clonal B-cell expansion in HIC and might serve as therapeutic targets in this debilitating disease. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Defining the activity of pro-reparative extracellular vesicles in wound healing based on miRNA payloads and cell type-specific lineage mapping.

Small extracellular vesicles (EVs) are released by cells and deliver biologically active payloads to coordinate the response of multiple cell types in cutaneous wound healing. Here we used a cutaneous injury model as a donor of pro-reparative EVs to treat recipient diabetic obese mice, a model of impaired wound healing. We established a functional screen for microRNAs (miRNAs) that increased the pro-reparative activity of EVs and identified a down-regulation of miR-425-5p in EVs in vivo and in vitro associated with the regulation of adiponectin. We tested a cell type-specific reporter of a tetraspanin CD9 fusion with GFP to lineage map the release of EVs from macrophages in the wound bed, based on the expression of miR-425-5p in macrophage-derived EVs and the abundance of macrophages in EV donor sites. Analysis of different promoters demonstrated that EV release under the control of a macrophage-specific promoter was most abundant and that these EVs were internalized by dermal fibroblasts. These findings suggested that pro-reparative EVs deliver miRNAs, such as miR-425-5p, that stimulate the expression of adiponectin that has insulin-sensitizing properties. We propose that EVs promote intercellular signaling between cell layers in the skin to resolve inflammation, induce proliferation of basal keratinocytes, and accelerate wound closure.

Antisense oligonucleotide-mediated disruption of HTT caspase-6 cleavage site ameliorates the phenotype of YAC128 Huntington disease mice.

In Huntington disease, cellular toxicity is particularly caused by toxic protein fragments generated from the mutant huntingtin (HTT) protein. By modifying the HTT protein, we aim to reduce proteolytic cleavage and ameliorate the consequences of mutant HTT without lowering total HTT levels. To that end, we use an antisense oligonucleotide (AON) that targets HTT pre-mRNA and induces partial skipping of exon 12, which contains the critical caspase-6 cleavage site. Here, we show that AON-treatment can partially restore the phenotype of YAC128 mice, a mouse model expressing the full-length human HTT gene including 128 CAG-repeats. Wild-type and YAC128 mice were treated intracerebroventricularly with AON12.1, scrambled AON or vehicle starting at 6 months of age and followed up to 12 months of age, when MRI was performed and mice were sacrificed. AON12.1 treatment induced around 40% exon skip and protein modification. The phenotype on body weight and activity, but not rotarod, was restored by AON treatment. Genes differentially expressed in YAC128 striatum changed toward wild-type levels and striatal volume was preserved upon AON12.1 treatment. However, scrambled AON also showed a restorative effect on gene expression and appeared to generally increase brain volume.

Impact of therapeutic inhibition of oncogenic cell signaling tyrosine kinase on cell metabolism: in vivo-detectable metabolic biomarkers of inhibition.

BACKGROUND: Inhibition of kinases is the ever-expanding therapeutic approach to various types of cancer. Typically, assessment of the treatment response is accomplished by standard, volumetric imaging procedures, performed weeks to months after the onset of treatment, given the predominantly cytostatic nature of the kinase inhibitors, at least when used as single agents. Therefore, there is a great clinical need to develop new monitoring approaches to detect the response to kinase inhibition much more promptly. Noninvasive 1H magnetic resonance spectroscopy (MRS) can measure in vitro and in vivo concentration of key metabolites which may potentially serve as biomarkers of response to kinase inhibition. METHODS: We employed mantle cell lymphoma (MCL) cell lines demonstrating markedly diverse sensitivity of inhibition of Bruton's tyrosine kinase (BTK) regarding their growth and studied in-depth effects of the inhibition on various aspects of cell metabolism including metabolite synthesis using metabolomics, glucose and oxidative metabolism by Seahorse XF technology, and concentration of index metabolites lactate, alanine, total choline and taurine by 1H MRS. RESULTS: Effective BTK inhibition profoundly suppressed key cell metabolic pathways, foremost pyrimidine and purine synthesis, the citrate (TCA) cycle, glycolysis, and pyruvate and glutamine/alanine metabolism. It also inhibited glycolysis and amino acid-related oxidative metabolism. Finally, it profoundly and quickly decreased concentration of lactate (a product of mainly glycolysis) and alanine (an indicator of amino acid metabolism) and, less universally total choline both in vitro and in vivo, in the MCL xenotransplant model. The decrease correlated directly with the degree of inhibition of lymphoma cell expansion and tumor growth. CONCLUSIONS: Our results indicate that BTK inhibition exerts a broad and profound suppressive effect on cell metabolism and that the affected index metabolites such as lactate, alanine may serve as early, sensitive, and reliable biomarkers of inhibition in lymphoma patients detectable by noninvasive MRS-based imaging method. This kind of imaging-based detection may also be applicable to other kinase inhibitors, as well as diverse lymphoid and non-lymphoid malignancies.

High expression of SRSF1 facilitates osteosarcoma progression and unveils its potential mechanisms.

BACKGROUND: SRSF1, a member of Serine/Arginine-Rich Splicing Factors (SRSFs), has been observed to significantly influence cancer progression. However, the precise role of SRSF1 in osteosarcoma (OS) remains unclear. This study aims to investigate the functions of SRSF1 and its underlying mechanism in OS. METHODS: SRSF1 expression level in OS was evaluated on the TCGA dataset, TAGET-OS database. qRT-PCR and Western blotting were employed to assess SRSF1 expression in human OS cell lines as well as the interfered ectopic expression states. The effect of SRSF1 on cell migration, invasion, proliferation, and apoptosis of OS cells were measured by transwell assay and flow cytometry. RNA sequence and bioinformatic analyses were conducted to elucidate the targeted genes, relevant biological pathways, and alternative splicing (AS) events regulated by SRSF1. RESULTS: SRSF1 expression was consistently upregulated in both OS samples and OS cell lines. Diminishing SRSF1 resulted in reduced proliferation, migration, and invasion and increased apoptosis in OS cells while overexpressing SRSF1 led to enhanced growth, migration, invasion, and decreased apoptosis. Mechanistically, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA) revealed that the biological functions of SRSF1 were closely associated with the dysregulation of the protein targeting processes, location of the cytosolic ribosome, extracellular matrix (ECM), and proteinaceous extracellular matrix, along with the PI3K-AKT pathway, Wnt pathway, and HIPPO pathway. Transcriptome analysis identified AS events modulated by SRSF1, especially (Skipped Exon) SE events and (Mutually exclusive Exons) MXE events, revealing potential roles of targeted molecules in mRNA surveillance, RNA degradation, and RNA transport during OS development. qRT-PCR confirmed that SRSF1 knockdown resulted in the occurrence of alternative splicing of SRRM2, DMKN, and SCAT1 in OS. CONCLUSIONS: Our results highlight the oncogenic role of high SRSF1 expression in promoting OS progression, and further explore the potential mechanisms of action. The significant involvement of SRSF1 in OS development suggests its potential utility as a therapeutic target in OS.

Single-cell RNA-seq with spatial transcriptomics to create an atlas of human diabetic kidney disease.

Diabetic kidney disease (DKD) develops in ~40% of patients with diabetes and is the leading cause of chronic kidney disease worldwide. We used single-cell RNA-sequencing and spatial transcriptomic analyses of kidney specimens from patients with DKD. Unsupervised clustering revealed distinct cell clusters, including epithelial cells and fibroblasts. We also identified differentially expressed genes (DEGs) and assessed enrichment, and cell-cell interactions. Specific enrichment of DKD was evident in venous endothelial cells (VECs) and fibroblasts with elevated CCL19 expression. The DEGs in most kidney parenchymal cells in DKD were primarily enriched in inflammatory signaling pathways. Intercellular crosstalk revealed that most cell interactions in DKD are associated with chemokines. Spatial transcriptomics revealed that VECs co-localized with fibroblasts, with most immune cells being enriched in areas of renal fibrosis. These results provided insight into the cell populations, intercellular interactions, and signaling pathways underlying the pathogenesis and potential targets for treating DKD.

The relationship between Sjögren's syndrome and recurrent pregnancy loss: a bioinformatics analysis.

RESEARCH QUESTION: As Sjögren's syndrome is an autoimmune disease and an essential factor in recurrent pregnancy loss (RPL), are there gene-related relationships between the pathogenesis of Sjögren's syndrome and RPL? DESIGN: The gene datasets for Sjögren's syndrome and RPL were obtained from the Gene Expression Omnibus database, and the co-expression modules and shared differentially expressed genes were identified through weighted gene co-expression network analysis (WGCNA) and limma analysis based on sample size. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analyses were applied to reveal the hidden biological pathways. Additionally, shared hub gene identification, gene set enrichment analysis, association of the hub gene with ferroptosis and immunity, drug sensitivity analysis, single-cell RNA sequencing analysis, and construction of the competing endogenous RNA (ceRNA) network were conducted. RESULTS: By intersecting the genes from WGCNA and limma analysis, one shared hub gene (KCNN3) was derived, exhibiting up-regulation in Sjögren's syndrome and RPL. There was a positive relationship between KCNN3 and the immune-related gene TLR2. The ceRNA network revealed that XIST was the most shared long non-coding RNA, which may bind competitively with eight microRNA to regulate the expression of KCNN3. Forty-eight drugs were found to be strongly associated with KCNN3 expression, including estramustine and cyclosporine. Moreover, KCNN3 exhibited high expression in RPL endothelial cells of villous tissue. CONCLUSIONS: This is one of the first studies to reveal that Sjögren's syndrome shares common biological pathways with RPL. KCNN3 was identified as the hub gene associated with Sjögren's syndrome and RPL, and may be a new target for mechanistic studies on Sjögren's syndrome and RPL.

Genomic landscape of glioblastoma without IDH somatic mutation in 42 cases: a comprehensive analysis using RNA sequencing data.

PURPOSE: Glioblastoma is a malignant brain tumor with a poor prognosis. Genetic mutations associated with this disease are complex are not fully understood and require further elucidation for the development of new treatments. The purpose of this study was to comprehensively analyze genetic mutations in glioblastomas and evaluate the usefulness of RNA sequencing. PATIENTS AND METHODS: We analyzed 42 glioblastoma specimens that were resected in routine clinical practice and found wild-type variants of the IDH1 and IDH2 genes. RNA was extracted from frozen specimens and sequenced, and genetic analyses were performed using the CLC Genomics Workbench. RESULTS: The most common genetic alterations in the 42 glioblastoma specimens were TP53 mutation (28.6%), EGFR splicing variant (16.7%), EGFR mutation (9.5%), and FGFR3 fusion (9.5%). Novel genetic mutations were detected in 8 patients (19%). In 12 cases (28.6%), driver gene mutations were not detected, suggesting an association with PPP1R14A overexpression. Our findings suggest the transcription factors SOX10 and NKX6-2 are potential markers in glioblastoma. CONCLUSION: RNA sequencing is a promising approach for genotyping glioblastomas because it provides comprehensive information on gene expression and is relatively cost-effective.

Analysis of the Expression of PRDX6 in Patients with Hepatocellular Carcinoma and its Effect on the Phenotype of Hepatocellular Carcinoma Cells.

Objectives: This research aimed to study the expression of PRDX6 mRNA in hepatocellular carcinoma (HCC) and its effect on the prognosis of HCC. Moreover, the effect of PRDX6 gene knockdown on the proliferation, migration, and invasion of HepG2 cells mediated by lentivirus was also examined. This study offers a theoretical and experimental basis for further research on the mechanism of PRDX6 in liver cancer and new methods for clinical diagnosis and treatment. Methods: RNA sequence data of 369 HCC patients were screened through the TCGA database, and the expression and clinical characteristics of PRDX6 mRNA were analyzed based on high-throughput RNA sequencing data. HepG2 cells were divided into WT, sh-NC and sh-PRDX6 groups. Real-time PCR and Western blot were used to detect the expression levels of the PRDX6 gene and protein, respectively. CCK8 method was used to detect the proliferation activity of HepG2 cells, scratch healing test was used to detect the migration ability, Transwell chamber was used to detect the invasion ability, and Western blot was used to detect the expression levels of PI3K/Akt/mTOR signaling pathway and Notch signaling pathway-related proteins. Results: The expression of PRDX6 was significantly correlated with the gender, race, clinical stage, histological grade, and survival time of HCC patients (P < 0.05). Compared with that in WT and sh-NC groups, the expression level of PRDX6 protein in HCC patients was significantly lower (P < 0.01), the proliferation activity of HCC cells was significantly decreased (P < 0.05), and the migration and invasion ability was significantly decreased (P < 0.05) in the sh-PRDX6 group. The expression levels of PI3K, p-Akt, p-mTOR, Notch1, and Hes1 proteins in the sh-PRDX6 group were significantly lower than those in WT and sh-NC groups (P < 0.05). Conclusion: The expression of PRDX6 may be closely related to the prognosis of HCC. Lentivirus-mediated PRDX6 knockdown can inhibit the proliferation, migration and invasion of HCC cells, which may be related to its regulating the PI3K/Akt/mTOR and Notch1 signaling pathways. PRDX6 is expected to be a new target for the diagnosis and treatment of liver cancer.

Activation of CD8+ T Cells in Chronic Obstructive Pulmonary Disease Lung.

Rationale: Despite the importance of inflammation in chronic obstructive pulmonary disease (COPD), the immune cell landscape in the lung tissue of patients with mild-moderate disease has not been well characterized at the single-cell and molecular level. Objectives: To define the immune cell landscape in lung tissue from patients with mild-moderate COPD at single-cell resolution. Methods: We performed single-cell transcriptomic, proteomic, and T-cell receptor repertoire analyses on lung tissue from patients with mild-moderate COPD (n = 5, Global Initiative for Chronic Obstructive Lung Disease I or II), emphysema without airflow obstruction (n = 5), end-stage COPD (n = 2), control (n = 6), or donors (n = 4). We validated in an independent patient cohort (N = 929) and integrated with the Hhip+/- murine model of COPD. Measurements and Main Results: Mild-moderate COPD lungs have increased abundance of two CD8+ T cell subpopulations: cytotoxic KLRG1+TIGIT+CX3CR1+ TEMRA (T effector memory CD45RA+) cells, and DNAM-1+CCR5+ T resident memory (TRM) cells. These CD8+ T cells interact with myeloid and alveolar type II cells via IFNG and have hyperexpanded T-cell receptor clonotypes. In an independent cohort, the CD8+KLRG1+ TEMRA cells are increased in mild-moderate COPD lung compared with control or end-stage COPD lung. Human CD8+KLRG1+ TEMRA cells are similar to CD8+ T cells driving inflammation in an aging-related murine model of COPD. Conclusions: CD8+ TEMRA cells are increased in mild-moderate COPD lung and may contribute to inflammation that precedes severe disease. Further study of these CD8+ T cells may have therapeutic implications for preventing severe COPD.

Increase in cathepsin K gene expression in Duchenne muscular dystrophy skeletal muscle.

Dystrophinopathy is caused by alterations in the dystrophin gene. The severe phenotype, Duchenne muscular dystrophy (DMD), is caused by a lack of dystrophin in skeletal muscles, resulting in necrosis and regenerating fibers, inflammatory cells, and muscle fibrosis. Progressive muscle weakness is a characteristic finding of this condition. Here, we encountered a rare case of a 10-year-old patient with asymptomatic dystrophinopathy with no dystrophin expression and investigated the reason for the absence of muscle weakness to obtain therapeutic insights for DMD. Using RNA-seq analysis, gene expression in skeletal muscles was compared among patients with asymptomatic dystrophinopathy, three patients with typical DMD, and two patients without dystrophinopathy who were leading normal daily lives. Cathepsin K (CTSK), myosin heavy chain 3 (MYH3), and nodal modulator 3-like genes exhibited a >8-fold change, whereas crystallin mu gene (CRYM) showed a <1/8-fold change in patients with typical DMD compared with their expression in the patient with asymptomatic dystrophinopathy. Additionally, CTSK and MYH3 expression exhibited a >16-fold change (P < 0.01), whereas CRYM expression showed a <1/16-fold change (P < 0.01) in patients with typical DMD compared with their expression in those without dystrophinopathy. CTSK plays an essential role in skeletal muscle loss, fibrosis, and inflammation in response to muscles injected with cardiotoxin, one of the most common reagents that induce muscle injury. Increased CTSK expression is associated with muscle injury or necrosis in patients with DMD. The lack of muscle weakness in the patient with asymptomatic dystrophinopathy might be attributed to the low CTSK expression in the muscles. To the best of our knowledge, this is the first report to demonstrate that CTSK expression was significantly higher in the skeletal muscles of patients with DMD with a typical phenotype than in those without dystrophinopathy.

Estimating the proportion of true null hypotheses and adaptive false discovery rate control in discrete paradigm.

Storey's estimator for the proportion of true null hypotheses, originally proposed under the continuous framework, has been modified in this work under the discrete framework. The modification results in improved estimation of the parameter of interest. The proposed estimator is used to formulate an adaptive version of the Benjamini-Hochberg procedure. Control over the false discovery rate by the proposed adaptive procedure has been proved analytically. The proposed estimate is also used to formulate an adaptive version of the Benjamini-Hochberg-Heyse procedure. Simulation experiments establish the conservative nature of this new adaptive procedure. Substantial amount of gain in power is observed for the new adaptive procedures over the standard procedures. For demonstration of the proposed method, two important real life gene expression data sets, one related to the study of HIV and the other related to methylation study, are used.

Genetic Analysis of Partially Resistant and Susceptible Chickpea Cultivars in Response to Ascochyta rabiei Infection.

The molecular mechanism involved in chickpea (Cicer arietinum L.) resistance to the necrotrophic fungal pathogen Ascochyta rabiei is not well documented. A. rabiei infection can cause severe damage in chickpea, resulting in significant economic losses. Understanding the resistance mechanism against ascochyta blight can help to define strategies to develop resistant cultivars. In this study, differentially expressed genes from two partially resistant cultivars (CDC Corinne and CDC Luna) and a susceptible cultivar (ICCV 96029) to ascochyta blight were identified in the early stages (24, 48 and 72 h) of A. rabiei infection using RNA-seq. Altogether, 3073 genes were differentially expressed in response to A. rabiei infection across different time points and cultivars. A larger number of differentially expressed genes (DEGs) were found in CDC Corinne and CDC Luna than in ICCV 96029. Various transcription factors including ERF, WRKY, bHLH and MYB were differentially expressed in response to A. rabiei infection. Genes involved in pathogen detection and immune signalings such as receptor-like kinases (RLKs), Leucine-Rich Repeat (LRR)-RLKs, and genes associated with the post-infection defence response were differentially expressed among the cultivars. GO functional enrichment and pathway analysis of the DEGs suggested that the biological processes such as metabolic process, response to stimulus and catalytic activity were overrepresented in both resistant and susceptible chickpea cultivars. The expression patterns of eight randomly selected genes revealed by RNA-seq were confirmed by quantitative PCR (qPCR) analysis. The results provide insights into the complex molecular mechanism of the chickpea defence in response to the A. rabiei infection.

Inhibitory Effects of Surface Pre-Reacted Glass Ionomer Filler Eluate on Streptococcus mutans in the Presence of Sucrose.

The surface pre-reacted glass ionomer (S-PRG) filler is a type of bioactive functional glass that releases six different ions. This study examined the effects of the S-PRG filler eluate on Streptococcus mutans in the presence of sucrose. In a solution containing S. mutans, the concentrations of BO33-, Al3+, Sr2+, and F- were significantly higher in the presence of the S-PRG filler eluate than in its absence (p < 0.001). The concentrations of these ions further increased in the presence of sucrose. Additionally, the S-PRG filler eluate significantly reduced glucan formation by S. mutans (p < 0.001) and significantly increased the pH of the bacterial suspension (p < 0.001). Bioinformatic analyses revealed that the S-PRG filler eluate downregulated genes involved in purine biosynthesis (purC, purF, purL, purM, and purN) and upregulated genes involved in osmotic pressure (opuAa and opuAb). At a low pH (5.0), the S-PRG filler eluate completely inhibited the growth of S. mutans in the presence of sucrose and significantly increased the osmotic pressure of the bacterial suspension compared with the control (p < 0.001). These findings suggest that ions released from the S-PRG filler induce gene expression changes and exert an inhibitory effect on S. mutans in the presence of sucrose.

Cell Heterogeneity and Variability in Peripheral Nerve after Injury.

With the development of single-cell sequencing technology, the cellular composition of more and more tissues is being elucidated. As the whole nervous system has been extensively studied, the cellular composition of the peripheral nerve has gradually been revealed. By summarizing the current sequencing data, we compile the heterogeneities of cells that have been reported in the peripheral nerves, mainly the sciatic nerve. The cellular variability of Schwann cells, fibroblasts, immune cells, and endothelial cells during development and disease has been discussed in this review. The discovery of the architecture of peripheral nerves after injury benefits the understanding of cellular complexity in the nervous system, as well as the construction of tissue engineering nerves for nerve repair and axon regeneration.

Profiling Molecular Changes of Host Response to Predict Outcome in Children with Septic Shock.

Background: Septic shock is associated with high mortality and there is significant heterogeneity in the host response. The aim of this study was to understand the genome-wide expression transcriptomic signatures in children with septic shock and correlate them with outcomes. Methods: This was a prospective study conducted on children (aged 1 month to 18 years) admitted to the PICU (June-December 2021) with septic shock. Demographic details, clinical details, and administered treatment were collected. Differential gene expression analysis was performed to understand the genes and pathways affecting in different subjects. Results: Fifteen patients were recruited (Septic shock survivors (n = 5), nonsurvivors (n = 5), and non-sepsis controls (n = 5). The median age of the patients in survivors and nonsurvivors was 15 (13, 24) months and 180 (180, 184) months, respectively. The sepsis-survivors vs nonsepsis possessed 983 upregulated and 624 downregulated genes while comparing sepsis nonsurvivors (SNS) with nonsepsis yielded 1,854 upregulated and 1,761 downregulated genes. Further, the lowest number of deregulated genes (383 upregulated and 486 downregulated) were present in SNS compared to sepsis survivors. The major Reactome pathways, found upregulated in SNSs relative to survivors included CD22 mediated B cell receptor (BCR) regulation, scavenging of heme from plasma, and creation of C4 and C2 activators while T cell receptor (TCR) signaling, the common pathway of fibrin clot formation and generation of second messenger molecules were found to be downregulated. Conclusion: Mortality-related gene signatures are promising diagnostic biomarkers for pediatric sepsis. How to cite this article: Lalitha AV, Vasudevan A, Moorthy M, Ramaswamy G. Profiling Molecular Changes of Host Response to Predict Outcome in Children with Septic Shock. Indian J Crit Care Med 2024;28(9):879-886.

Classification and characterisation of extracellular vesicles-related tuberculosis subgroups and immune cell profiles.

Around the world, tuberculosis (TB) remains one of the most common causes of morbidity and mortality. The molecular mechanism of Mycobacterium tuberculosis (Mtb) infection is still unclear. Extracellular vesicles (EVs) play a key role in the onset and progression of many disease states and can serve as effective biomarkers or therapeutic targets for the identification and treatment of TB patients. We analysed the expression profile to better clarify the EVs characteristics of TB and explored potential diagnostic markers to distinguish TB from healthy control (HC). Twenty EVs-related differentially expressed genes (DEGs) were identified, and 17 EVs-related DEGs were up-regulated and three DEGs were down-regulated in TB samples, which were related to immune cells. Using machine learning, a nine EVs-related gene signature was identified and two EVs-related subclusters were defined. The single-cell RNA sequence (scRNA-seq) analysis further confirmed that these hub genes might play important roles in TB pathogenesis. The nine EVs-related hub genes had excellent diagnostic values and accurately estimated TB progression. TB's high-risk group had significantly enriched immune-related pathways, and there were substantial variations in immunity across different groups. Furthermore, five potential drugs were predicted for TB using CMap database. Based on the EVs-related gene signature, the TB risk model was established through a comprehensive analysis of different EV patterns, which can accurately predict TB. These genes could be used as novel biomarkers to distinguish TB from HC. These findings lay the foundation for further research and design of new therapeutic interventions aimed at treating this deadly infectious disease.

Single-cell transcriptome analysis reveals endometrial immune microenvironment in minimal/mild endometriosis.

Endometriosis is a common inflammatory disorder in women of reproductive age due to an abnormal endometrial immune environment and is associated with infertility. This study aimed to systematically understand the endometrial leukocyte types, inflammatory environment, and impaired receptivity at single-cell resolution. We profiled single-cell RNA transcriptomes of 138 057 endometrial cells from endometriosis patients (n = 6) and control (n = 7), respectively, using 10x Genomics platform. We found that one cluster of epithelial cells that expressed PAEP and CXCL14 was mostly from the control during the window of implantation (WOI). This epithelial cell type is absent in the eutopic endometrium during the secretory phase. The proportion of endometrial immune cells decreased in the secretory phase in the control group, whereas the cycle variation of total immune cells, NK cells, and T cells was absent in endometriosis. Endometrial immune cells secreted more IL-10 in the secretory phase than in the proliferative phase in the control group; the opposite trend was observed in endometriosis. Proinflammatory cytokines levels in the endometrial immune cells were higher in endometriosis than in the control group. Trajectory analysis revealed that the secretory phase epithelial cells decreased in endometriosis. Ligand-receptor analysis revealed that 11 ligand-receptor pairs were upregulated between endometrial immune and epithelial cells during WOI. These results provide new insights into the endometrial immune microenvironment and impaired endometrial receptivity in infertile women with minimal/mild endometriosis.

Sequence characterization of extracellular HBV RNA in patient plasma.

Antiviral nucleos(t)ide analogue therapies inhibit HBV replication and suppress the HBV DNA levels in patients with chronic HBV infection. Since HBV RNAs are expressed from cccDNA or HBV integrated sequences, independently of viral genome replication, levels of HBV RNAs in plasma may remain high following treatment with nucleos(t)ide analogue. Thus, HBV RNAs have been proposed to be used as a viral biomarker for treatment outcome and disease progression. Recent investigations of plasma HBV RNAs described the presence of full length as well as subgenomic forms of RNA. To support the usage of plasma HBV RNAs as a viral biomarker, further understanding of HBV RNA composition in clinical samples is needed. Here, sequence of extracellular HBV RNAs was characterized in plasma samples of patients with chronic HBV infection using two independent RNA amplification methods that do not use HBV-specific primers for amplification: total RNA (NuGEN RNAseq) and mRNA (TruSeq RNAseq). Sequencing coverage was obtained across the full length of HBV genome for both methods, confirming the presence of full-length HBV RNA in plasma. The sequence of HBV RNA was nearly identical to plasma HBV DNA sequence in each sample with only 0-14 (median 4) mismatches over 3 kb. Thus, sequence of HBV RNA plasma reflects the intrahepatic viral reservoir and can be used for monitoring of sequence variants such as resistance in clinical trials. Additionally, RNA splice forms, different polyA tails start positions and presence of HBV-human chimeric transcript were identified.