JOURNAL/nrgr/04.03/01300535-202512000-00030/figure1/v/2025-01-31T122243Z/r/image-tiff Studies have shown that vascular dysfunction is closely related to the pathogenesis of Alzheimer's disease. The middle temporal gyrus region of the brain is susceptible to pronounced impairment in Alzheimer's disease. Identification of the molecules involved in vascular aberrance of the middle temporal gyrus would support elucidation of the mechanisms underlying Alzheimer's disease and discovery of novel targets for intervention. We carried out single-cell transcriptomic analysis of the middle temporal gyrus in the brains of patients with Alzheimer's disease and healthy controls, revealing obvious changes in vascular function. CellChat analysis of intercellular communication in the middle temporal gyrus showed that the number of cell interactions in this region was decreased in Alzheimer's disease patients, with altered intercellular communication of endothelial cells and pericytes being the most prominent. Differentially expressed genes were also identified. Using the CellChat results, AUCell evaluation of the pathway activity of specific cells showed that the obvious changes in vascular function in the middle temporal gyrus in Alzheimer's disease were directly related to changes in the vascular endothelial growth factor (VEGF)A-VEGF receptor (VEGFR) 2 pathway. AUCell analysis identified subtypes of endothelial cells and pericytes directly related to VEGFA-VEGFR2 pathway activity. Two subtypes of middle temporal gyrus cells showed significant alteration in AD: endothelial cells with high expression of Erb-B2 receptor tyrosine kinase 4 (ERBB4 high ) and pericytes with high expression of angiopoietin-like 4 (ANGPTL4 high ). Finally, combining bulk RNA sequencing data and two machine learning algorithms (least absolute shrinkage and selection operator and random forest), four characteristic Alzheimer's disease feature genes were identified: somatostatin ( SST ), protein tyrosine phosphatase non-receptor type 3 ( PTPN3 ), glutinase ( GL3 ), and tropomyosin 3 ( PTM3 ). These genes were downregulated in the middle temporal gyrus of patients with Alzheimer's disease and may be used to target the VEGF pathway. Alzheimer's disease mouse models demonstrated consistent altered expression of these genes in the middle temporal gyrus. In conclusion, this study detected changes in intercellular communication between endothelial cells and pericytes in the middle temporal gyrus and identified four novel feature genes related to middle temporal gyrus and vascular functioning in patients with Alzheimer's disease. These findings contribute to a deeper understanding of the molecular mechanisms underlying Alzheimer's disease and present novel treatment targets.

Publicly accessible expression data produced by large consortium projects like TCGA and GTEx are increasing in number and size at an unprecedented rate. Their utility cannot be underestimated given the diversity of valuable tools widely used to interrogate these data and the many discoveries of biological and clinical significance already garnered from these datasets. However, there remain undiscovered ways to mine these rich resources and a continuing need to provide researchers with easily accessible and user-friendly applications for complex or bespoke analyses. We introduce TRanscriptome ANalysis of StratifiEd CohorTs (TRANSECT), a bioinformatics application automating the stratification and subsequent differential expression analysis of cohort data to provide further insights into gene regulation. TRANSECT works by defining two groups within a cohort based on disparate expression of a gene or a gene set and subsequently compares the groups for differences in global expression. Akin to reverse genetics minus the inherent requirement of in vitro or in vivo perturbations, cell lines or model organisms and all the while working within natural physiological limits of expression, TRANSECT compiles information about global transcriptomic change and functional outcomes. TRANSECT is freely available as a command line application or online at https://transect.au.

Tendon injury is a common and challenging problem in the motor system that lacks an effective treatment, affecting daily activities and lowering the quality of life. Limited tendon regenerative capability and immune microenvironment dyshomeostasis are considered the leading causes hindering tendon repair. The chirality of biomaterials was proved to dictate immune microenvironment and dramatically affect tissue repair. Herein, chiral hierarchical structure hydroxylapatite (CHAP) nanoplates are innovatively synthesized for immunomodulatory purposes and further coated onto polylactic acid electrospinning membranes to achieve long-term release for tendon regeneration adaption. Notably, levorotatory-chiral HAP (L-CHAP) nanoplates rather than dextral-chiral or racemic-chiral exhibit good biocompatibility and bioactivity. In vitro experiments demonstrate that L-CHAP induces macrophage M2 polarization by enhancing macrophage efferocytosis, which alleviates inflammatory damage to tendon stem cells (TDSCs) through downregulated IL-17-NF-κB signaling. Meanwhile, L-CHAP-mediated macrophage efferocytosis also promotes TDSCs proliferation and tenogenic differentiation. By establishing a rat model of Achilles tendon injury, L-CHAP was demonstrated to comprehensively promoting tendon repair by enhancing macrophage efferocytosis and M2 polarization in vivo, finally leading to improvement of tendon ultrastructural and mechanical properties and motor function. This novel strategy highlights the role of L-CHAP in tendon repair and thus provides a promising therapeutic strategy for tendon injury.

Altered lipid metabolism is a well-known hallmark of cancer. However, the underlying mechanisms of altered lipid metabolism in colorectal cancer (CRC) progression requires further investigation. Previously we have revealed that DEAD-box RNA helicase 21 (DDX21) promotes CRC metastasis via liquid-liquid phase separation. In this study, we identify DDX21 as a novel regulator of lipid metabolism in CRC.

In vitro and in vivo assays illustrated the biological role of DDX21 and YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in CRC lipid metabolism and progression. Bioinformatics analysis, ChIP, meRIP, RIP, RNA stability assay and dual-luciferase reporter assay were applied to explore the underlying molecular mechanisms. The expression levels and prognostic role of YTHDF1/DDX21/HMGCS1 axis in CRC patients were analyzed by immunohistochemical staining and Kaplan-Meier plotter.

DDX21 enhanced CRC progression via promoting lipid metabolism. Mechanistically, YTHDF1 enhanced DDX21 mRNA stability by recognizing its m6A-modified sites. DDX21 further binded to 3‑hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) promoter region and directly activated HMGCS1 transcription. Moreover, our clinical data showed that a simultaneously high expression of YTHDF1, DDX21 and HMGCS1 predicted an unfavorable overall survival in CRC patients.

Our study demonstrates that the YTHDF1/DDX21/HMGCS1 axis promotes CRC progression via regulating lipid metabolism and DDX21 might be a promising target for CRC therapy.

Liver hepatocellular carcinoma (LIHC) is a common cancer with poor prognosis. The FAM72 gene family enhances neuronal self-renewal, potentially increasing tumor formation, but its functional and predictive relevance in LIHC remains unclear. We sought to investigate the function of the FAM72 gene family in LIHC in the present study.

We acquired TCGA-LIHC expression and phenotypic data as well as extensive clinicopathologic information from the UCSC Xena Database (https://xenabrowser.net/datapages/) database. We analyzed the association between FAM72 gene family expression in LIHC and patient prognosis and immune infiltration; Genomic and functional enrichment analysis for FAM72 genes was analyzed. Finally, Western blot method, quantitative real-time polymerase chain reaction and CCK8 detection and cell invasion experiments were used to verify the effect of FAM72A expression on LIHC.

The expression of FAM72 gene family is different between LIHC and normal liver tissues. The expression of FAM72 gene family increased with increasing grading of LIHC tissues. The expression of FAM72 gene family was significantly reduced in LIHC stage IV. LIHC tissues expressed significantly more FAM72 genes than did normal tissues at the T stage (p < 0.001,). which has a good value in the diagnosis of LIHC (AUC greater than 0.85), and was strongly linked with the tumor stage in LIHC. Based on Cox analysis of univariate data, the FAM72 gene family was associated with poor overall survival (OS) in patients with LIHC. Analysis of multifactorial Cox data revealed an independent relationship between FAM72 expression and OS. Increased FAM72 gene expression is associated with poor survival rates and immune cell infiltration. Methylation levels were associated with the prognosis of patients with LIHC. Ultimately, our findings revealed that FAM72A is abundantly expressed in LIHC cells, facilitating proliferation and metastasis.

These findings indicate that the FAM72 gene family is a potential molecular marker for poor prognosis in LIHC, providing additional insights into the development of therapeutic approaches and prognostic markers.

Spinal cord injury (SCI) is a refractory diseases that leads to lasting impairments in sensory and motor functions. Following nerve injury, CX3CL1, a neuron membrane protein, is released. Research suggests that CX3CL1 neurons possess a unique signaling mechanism for rescue. The objective of this study was to investigate the neuroprotective mechanism of CX3CL1 after spinal cord injury. Our findings indicate that injured neurons release CX3CL1, which binds to microglia and activates the NF-κB pathway. This activation downregulates IKB and P65 phosphorylation, thereby controlling the progression of inflammation after spinal cord injury. Consequently, more neurons are protected, leading to the restoration of nerve function. In conclusion, this study has discovered that CX3CL1 played a critical role in neuroprotection following spinal cord injury, making it a potential therapeutic target for treating this condition.

Despite over a century of clinical electrocardiographic studies showing that females exhibit a faster resting heart rate (HR), the mechanisms underlying sex differences in HR remain unresolved. Moreover, inappropriate sinus tachycardia primarily affects females, whereas males are at a higher risk for conduction block and atrial fibrillation. We hypothesized that the sexual dimorphism of genes responsible for sinoatrial node (SAN) pacemaking and signaling pathways may contribute to the sex differences in HR and susceptibility to arrhythmias.

Human SAN central pacemaker and right atrial tissue were isolated from nondiseased ex vivo donor hearts. Gene expressions were quantified and validated using the transcriptomic panel and quantitative polymerase chain reaction. Gene set enrichment analysis, Ingenuity Pathway Analysis, and human-specific SAN models were utilized to define regulatory mechanisms and functional impacts of sex-biased gene transcription.

We identified differentially expressed region- and sex-specific genes, with gene sets enriched in HR regulation (eg, TBX3, HCN1) and metabolism (eg, ADIPOQ, LEP) pathways in female SAN. In contrast, differential genes and gene sets involved in collagen biosynthetic processes, fibrogenesis (eg, EGR1), and immune response (eg, IL6, CXCL8) pathways were enriched in males SAN and right atrial. Ingenuity Pathway Analysis predicted significant roles for TBX3 and estradiol in the sex-specific expression of genes involved in SAN function. Computational simulations showed that the sex-specific SAN differences in If (HCN1) and ICa,L (CACNA1D) can explain the faster HR in females, with females having a lower threshold for inappropriate sinus tachycardia, whereas males are more vulnerable to sinus arrest.

The human SAN exhibits region-specific sexual dimorphism in pacemaking gene sets. Higher expression of TBX3 and HCN1 in females may underlie their faster HR and increased susceptibility to inappropriate sinus tachycardia, whereas enriched gene sets related to inflammation and collagen biosynthesis in males may predispose them to conduction impairments and atrial fibrillation risk.

Interferon gamma (IFNγ) can amplify immune cell-mediated anti-tumor immunity, as well as directly kill cancer cells. Ginsenoside Rh2 (Rh2), a bioactive compound in traditional Chinese medicine, exhibits anti-cancer effects such as inhibiting proliferation and metastasis. Our earlier research found that Rh2 combined with IFNγ enhanced CXCL10 secretion in cancer cells. Here, we explored whether Rh2 and IFNγ exerted more potent anti-cancer activity in vitro and in vivo, along with its mechanisms and clinical value. Our data showed that Rh2 in combination with IFNγ resulted in a remarkably increased cytotoxicity in colorectal cancer cells including HT29, LoVo and T84 cell lines. Consistently, intratumoral injection with Rh2 plus IFNγ further restricted the HT29 tumor growth in vivo, and importantly, it was demonstrated to be safe for mice. Meanwhile, the combo treatment activated the stimulator of interferon genes (STING) pathway in cancer cells, promoting the transcription of downstream type I interferon. RNA sequencing revealed a dramatically transcriptional alteration in cancer cells with combo treatment and indicated that Rh2 further augmented the activation of interferon signaling pathway, compared with the IFNγ alone. Inhibition of janus kinase (JAK) by ruxolitinib could significantly rescue the cell death-triggered by the combo treatment. Then, a gene set named Rh2+IFNγ signature genes (RISG) was defined, which contained top 20 significantly upregulated genes from the combo treatment. Patients who exhibited a favorable response to the immunotherapy had a higher expression of RISG in tumor compared with those who did not respond. And the high expression of RISG was correlated with better clinical outcome in patients with colorectal cancer (CRC) and skin cutaneous melanoma (SKCM). Herein, the combination of Rh2 with IFNγ served as a promising strategy for cancer treatment, and its-derived RISG gene set also exhibited potential value in predicting clinical outcome. Schematic diagram of the anti-cancer effect of Rh2 combined with IFNγ. The schematic diagram illustrated that ginsenoside Rh2 in combination with IFNγ robustly activated the interferon signals in cancer cells, ultimately leading a significant cell death of cancer cells. ISGs, interferon-stimulated genes. Created with BioRender.com.

This retrospective study measured global gene abundance using RNASeq of blastocoel fluid-conditioned media from euploid ICSI-generated embryos to identify genes and signaling pathways associated with maternal age.

Blastocoel fluid-conditioned media was obtained following trophectoderm biopsy of ICSI-generated day-5 blastocysts. Media for RNASeq were from 24 euploid blastocysts (9 from patients aged 35 or older). Transcriptome analysis identified differentially expressed genes when comparing media from patients of advanced maternal age to those younger than 35. Further gene abundance analysis on genes and pathways identified from the RNASeq analysis was conducted with another group of media samples using RT-qPCR.

Twenty-five protein encoding genes identified in the RNASeq study were differentially expressed when comparing blastocoel fluid-conditioned media associated with patients of advanced maternal age to media associated with patients under the age of 35. Genes encoding the proteins SHARPIN and BCL2L12 showed a statistically significant increase (p < 0.05) in abundance in patients of advanced maternal age. Abundance analysis using RT-qPCR in additional media samples revealed elevated SHARPIN abundance in media associated with successful implantation in patients under 35 alongside a decrease in CASP8 abundance. This abundance pattern was the opposite in media associated with successful implantation in patients of advanced maternal age.

This study uncovered differential apoptotic gene abundance associated with maternal age by assessing blastocoel fluid-conditioned media from euploid blastocysts. These unique abundance patterns may provide insight into the regulation of apoptosis in embryos from women of advanced maternal age, and how this signaling pathway may impact implantation outcomes.

Cytokine licensing with pro-inflammatory molecules, such as tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), has emerged as a promising strategy to enhance the therapeutic potential of multipotent mesenchymal stromal cells (MSCs). While licensing has demonstrated benefits for immunomodulation, its effects on other key MSC functions, including differentiation and paracrine activity, remain incompletely explored. In this study, we evaluated the transcriptomic, metabolomic, and functional changes induced by short-term TNF-α/IFN-γ priming of Wharton's jelly-derived MSCs (WJ-MSCs).

WJ-MSCs were expanded and exposed to TNF-α and IFN-γ (10 ng/ml each) for 24 h. Transcriptomic analysis was performed using RNA sequencing to identify differentially expressed genes related to immune modulation and lineage commitment. Metabolomic profiling was conducted using high-resolution mass spectrometry to assess changes in metabolic pathways. Functional assays evaluated the effects of cytokine priming on induced differentiation and growth factor secretion.

Cytokine licensing induced notable alterations in gene expression, upregulating pathways linked to immune response, inflammation, and cytokine signalling. However, short-term cytokine treatment significantly attenuated the osteogenic and adipogenic differentiation of MSCs, as evidenced by the reduced expression of RUNX2, ALP, CEBPA, and PPARG. The priming had a negligible effect on EGF, FGF-2, HGF, LIF, and SCF secretion. The production of VEGF-A and VEGF-C was elevated, although the levels remained low. Metabolomic analysis revealed enhanced kynurenine pathway activity, indicative of increased tryptophan catabolism, accompanied by elevated levels of fatty acids and polyamines.

Our findings demonstrate that TNF-α/IFN-γ priming reprograms WJ-MSCs by enhancing their immunomodulatory capacity at the expense of differentiation potential. These results highlight the need for tailored strategies to optimize MSC functionality for specific clinical applications.

Arginine auxotrophy has been reported in a subset of cancers with inherently defective de novo arginine synthesis. However, the use of arginine deprivation therapy seems to be unequally effective, partially owing to the resistance acquired by cancer cells. Study of underlying factors involved in this response thus becomes of utmost importance. Meanwhile, the function of etoposide-induced 2.4 homolog (EI24) in cancer metabolism, and specifically in arginine metabolism, remains unknown.

EI24 was overexpressed in cancer cells using a doxycycline-inducible system or adenovirus transduction, while siRNA was used to knockdown EI24. Amino acid(s) deprivation medium was exploited with a cell viability assay to check the reliance of cancer cell survival on arginine. Protein expression and activation were examined through western blot and co-immunoprecipitation blot. Furthermore, global and specific protein translation were assessed through the SUnSET assay and polysome fractionation analysis. Gene expression and arginine level were downloaded from public cancer datasets for in silico validation including gene set enrichment and survival analysis to objectively evaluate the association between EI24 and arginine metabolism.

EI24 promoted cancer survival under arginine starvation. Mechanistically, EI24 replenished translation of argininosuccinate synthase 1 (ASS1) by inducing the inactive S-nitrosylated form of phosphatase and tensin homolog (PTEN), leading to release of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) axis. This tumor-promoting action of EI24 could be found in multiple ASS1-deficient cancer cells regardless of p53 status. Furthermore, expression of EI24 was linked to enrichment of arginine metabolism pathway as well as poor survival of patients with cancer across various cancer types, suggesting its role in cancer resistance to arginine deprivation.

This study is the first to report the role of EI24 in promoting cancer survival via translational regulation of the metabolic enzyme ASS1, thus paving a route for further investigation into the link between EI24 and cancer metabolism.

High-risk neuroblastoma frequently exhibits segmental gain of chromosome 17q, including the locus of PPM1D, which encodes the phosphatase WIP1, a regulator of p53 activity, DNA repair, and apoptosis. High expression of PPM1D is correlated to poor prognosis, and genetic or pharmacologic inhibition of WIP1 suppresses neuroblastoma growth. Here, we show that combining drugs that target WIP1 and H3K27 demethylation induces synergistic cytotoxicity in neuroblastoma. We screened 527 different compounds together with inhibitors of WIP1 and identified a strong cytotoxic synergism between the WIP1 inhibitor SL-176 and GSK-J4, a specific inhibitor of the H3K27 demethylase JMJD3. Viability assays in neuroblastoma cell lines and treatment of tumor spheroids confirmed the synergistic effect of combining SL-176 with GSK-J4. Immunoblot experiments demonstrated a marked effect on WIP1 downstream targets and apoptosis markers, while qPCR showed a synergistic upregulation of p53 downstream targets PUMA and p21. RNA sequencing revealed a vast number of differentially expressed genes, suggesting a pervasive effect of this drug combination on transcription, with enrichment of pathways involved in DNA damage response. Finally, this drug combination was confirmed to reduce tumor growth in zebrafish xenograft experiments. In conclusion, the combination of the WIP1 inhibitor SL-176 and the epigenetic modifier GSK-J4 induces synergistic cytotoxicity in neuroblastoma cells by potentiating p53 downstream effects.

Clear cell renal cell carcinoma (ccRCC) accounts for 70% of renal cell carcinoma (RCC) cases. Although surgery remains the mainstay treatment, renal injury and high metastasis rates after nephrectomy dramatically reduce patient quality of life. Drugs that stimulate the immune system by targeting checkpoint pathways improve overall survival in patients with RCC. Here, we investigated the applicability of lysophosphatidylcholine acyltransferase 3 (LPCAT3) as a target for immunotherapy.

In the present study, high LPCAT3 expression in ccRCC was identified using The Cancer Genome Atlas (TCGA) data and validated in two external cohorts from the Gene Expression Omnibus (GEO) database. qRT-PCR was performed to identify the mRNA level of LPCAT3 in tumors and adjacent normal tissues. And immunohistochemistry was used to evaluate the protein level of LPCAT3 between two groups of samples. Furthermore, gene set enrichment analysis was performed to explore the biological processes and pathways related to LPCAT3 expression. Key gene expression and correlation analyses were performed to determine the crosstalk among LPCAT3 expression, ferroptosis, and endoplasmic reticulum stress (ERS). Subsequently, CIBERSORT was used to analyze the immune infiltration status of patients with high and low LPCAT3 expression.

TCGA and GEO data revealed that LPCAT3 expression in ccRCC tumor tissues was higher than that in adjacent normal tissues; moreover, patients with high LPCAT3 expression had better survival outcomes. qRT-PCR and immunohistochemistry verified the high LPCAT3 expression in tumor tissue. Pathways related to ferroptosis and ERS were upregulated in patients with high LPCAT3 expression. Univariate and multivariate regression analyses revealed that low LPCAT3 levels represent an independent risk factor for ccRCC. LPCAT3 expression was positively correlated with M2 macrophage infiltration levels but negatively correlated with the memory B cell, CD8+ T cell, follicular helper T cell, regulatory T cell, activated natural killer cell, and activated memory CD4+ T cell infiltration levels.

LPCAT3was identified as a ccRCC biomarker and may regulate immune infiltration and prognosis in ccRCC by mediating ferroptosis and ERS. Thus, it has potential for exploitation as a prognostic and immune therapeutic target for patients with ccRCC.

Radiation pneumonitis (RP) is a common and severe complication of radiotherapy, whose pathogenesis involves complex inflammatory responses and cellular damage. Despite its clinical significance, effective treatments remain limited. This study investigates the role of radiation-induced PINK1/PRKN-mediated mitophagy and type I interferon responses in RP and evaluates the therapeutic potential of Urolithin A (UA) in regulating inflammation through mitophagy activation.

We established RP mouse models (20 Gy thoracic irradiation) and radiation-induced BEAS-2B cell models (6 Gy). We systematically investigated mitochondrial damage, mtRNA release, RIG-I/MDA5-MAVS pathway activation, and PINK1/PRKN-mediated mitophagy changes. Moreover, the effects of UA and the mitophagy inhibitor Mdivi-1 on inflammation and lung injury were analyzed.

Radiation significantly caused mitochondrial damage in lung tissues, inducing mtRNA release and RIG-I/MDA5-MAVS-mediated type I interferon response. PINK1/PRKN-mediated mitophagy was significantly enhanced, clearing damaged mitochondria and reducing cytosolic mtRNA release, thereby suppressing inflammation. Pharmacological activation of mitophagy with UA markedly improved lung pathology, reduced inflammatory cytokine levels, and inhibited excessive activation of the RIG-I/MDA5-MAVS pathway. Conversely, the knockdown of PINK1 or PRKN weakened the protective effects of UA. Both in vitro and in vivo, UA reduced radiation-induced inflammation and improved lung tissue structure and function through mitophagy.

Radiation-induced mtRNA release activates the RIG-I/MDA5-MAVS-mediated type I interferon response, driving inflammation in RP. PINK1/PRKN-mediated mitophagy significantly alleviates inflammation by reducing cytosolic mtRNA release. As a mitophagy inducer, UA demonstrates therapeutic potential for RP, providing a new direction for the development of anti-inflammatory strategies.

MYC is a master regulatory transcription factor whose sustained dysregulation promotes the initiation and maintenance of numerous cancers. While MYC is a regarded as a potenial therapeutic target in cancer, its intrinsically disordered structure has proven to be a formidable barrier toward the development of highly effective small molecule inhibitors. We rationalized that proteolysis targeting chimeras (PROTACs), which might accomplish the targeted degradation of MYC, would achieve more potent cell killing in MYC-driven cancer cells than reversible inhibitors. PROTACs are bifunctional small molecules designed to produce a ternary complex between a target protein and an E3 ligase leading the target's ubiquitination and degradation by the 26S proteasome. We generated PROTAC MTP3 based on modifications of the previously reported MYC-targeting compound KJ-Pyr-9. We found that MTP3 depletes endogenous full-length MYC proteins and uniquely induces increasing levels of a functional, N-terminally truncated MYC species, tMYC. Furthermore, MTP3 perturbs cellular MYC levels in favor of a tMYC-dominated state whose gene regulatory landscape is not significantly altered compared to that of wild type MYC. Moreover, although it lacks ∼10 kDa of MYC's N-terminal transactivation domain, tMYC is sufficient to maintain an oncogenic proliferative state. Our results highlight the complexities of proximity-inducing compounds against highly regulated and conformationally dynamic protein targets such as MYC and indicate that PROTACs can induce alternative outcomes beyond target protein degradation.

Age-associated B cells (ABCs) with autoreactive properties accumulate with age and expand prematurely in autoimmune diseases. However, the mechanisms behind ABC generation and maintenance remain poorly understood. We show that continuous B cell receptor (BCR) signaling is essential for ABC development from anergic B cells in aged and autoimmune mice. ABCs exhibit constitutive BCR activation, with surface BCRs being internalized. Notably, anergic B cells, but not nonautoreactive B cells, contributed to ABC formation in these models. Anergic B cells also showed a greater propensity for in vitro differentiation into ABCs, which was inhibited by the expression of the transcription factor Nr4a1. Bruton's tyrosine kinase (Btk), a key BCR signaling component, was constitutively activated in ABCs from aged and autoimmune mice as well as patients with lupus. Inhibiting Btk reduced ABC numbers and ameliorated the pathogenicity of lupus mice. Our findings reveal critical mechanisms underlying ABC development and offer previously unrecognized therapeutic insights for autoimmune diseases.

Circular RNA (circRNA) is a class of noncoding RNA with regulatory potentials. Its role in the transdifferentiation of prostate and lung adenocarcinoma into neuroendocrine prostate cancer (NEPC) and small cell lung cancer (SCLC) remains unexplored. Here, we identified circRMST as an exceptionally abundant circRNA predominantly expressed in NEPC and SCLC, with strong conservation between humans and mice. Functional studies using shRNA, siRNA, CRISPR-Cas13, and Cas9 consistently demonstrate that circRMST is essential for tumor growth and the expression of ASCL1, a master regulator of neuroendocrine fate. Genetic knockout of Rmst in NEPC genetic engineered mouse models prevents neuroendocrine transdifferentiation, maintaining tumors in an adenocarcinoma state. Mechanistically, circRMST physically interacts with lineage transcription factors NKX2-1 and SOX2. Loss of circRMST induces NKX2-1 protein degradation through autophagy-lysosomal pathway and alters the genomic binding of SOX2, collectively leading to the loss of ASCL1 transcription.

CDK9 coordinates signaling events that regulate transcription and is implicated in oncogenic pathways, making it an actionable target for drug development. While numerous CDK9 inhibitors have been developed, success in the clinic has been limited. Targeted degradation offers a promising alternative. A comprehensive evaluation of degradation versus inhibition is needed to assess when degradation might offer superior therapeutic outcomes. We report a selective and potent CDK9 degrader with rapid kinetics, comparing its downstream effects to those of a conventional inhibitor. We validated that CDK9 inhibition triggers a compensatory feedback mechanism that dampens its anticipated effect on MYC expression and found that this was absent when degraded. Importantly, degradation is more effective at disrupting MYC transcriptional regulation and subsequently destabilizing nucleolar homeostasis, likely by abrogation of both enzymatic and scaffolding functions of CDK9. These findings suggest that CDK9 degradation offers a more robust strategy to overcome limitations associated with its inhibition.

Enhanced glycolytic levels in cancer cells are a common characteristic of many cancer types. Modulation of glycolytic metabolism is crucial for enhancing the efficacy of cancer therapy. The specific role of 6-methoxyflavone in regulating glycolytic metabolism in cancer cells remains unclear. This study aimed to elucidate the impact of 6-methoxyflavone on glycolytic metabolism in cervical cancer cells and its clinical relevance.

The tandem mass tag (TMT) proteomic analysis was used to identify significantly enriched biological processes and pathways in HeLa cells after treatment with 6-methoxyflavone. Additionally, the differential expression of glycolysis-related proteins was validated using parallel reaction monitoring (PRM) proteomics. Untargeted and targeted metabolomics analyses were used to identify differentially expressed glycolysis-related metabolites. Furthermore, alternative splicing, new transcripts, and domain analyses were used to detect the effects of 6-methoxyflavone on the structures of glycolysis-related genes and proteins. Subcellular localization, molecular docking, and non-covalent interaction analyses were used to detect the subcellular localization, affinity of 6-methoxyflavone for glycolysis-related proteins, and sites of non-covalent interactions. Clinical characteristics and immunological correlation analyses were used to elucidate the relationships between glycolysis-related genes and clinicopathological characteristics, survival, prognosis, and immune-related indicators of patients with cervical cancer. Finally, glycolysis stress tests and enzyme activity assays were used to verify the effect of 6-methoxyflavone on glycolysis in HeLa cells.

TMT and PRM proteomics, as well as untargeted and targeted metabolomics results, showed that 6-methoxyflavone downregulated the expression levels of glycolysis-related proteins and metabolites in HeLa cells, and that the structures and functions of glycolysis-related genes and proteins in the cytoplasm underwent changes. 6-Methoxyflavone had a good affinity for nine glycolysis-related proteins, all of which had non-covalent interaction sites. Clinical characteristics and immune correlation analyses showed relationships between 6-methoxyflavone and five clinical characteristics, survival prognosis, and four immune-related indicators in patients with cervical cancer. After treatment with 6-methoxyflavone, the basal glycolytic level, maximum glycolytic capacity, and glycolytic reserve of HeLa cells were downregulated. Additionally, 6-methoxyflavone inhibited the activity of pyruvate kinase.

6-Methoxyflavone inhibited energy metabolism in HeLa cells through the glycolysis pathway. 6-Methoxyflavone may be related to five clinical characteristics, prognosis, tumor microenvironment, immune cells, immune checkpoints, and immunotherapy efficacy in patients with cervical cancer.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. The primary causes of COPD are environmental, including cigarette smoking; however, genetic susceptibility also contributes to COPD risk. Genome-Wide Association Studies (GWASes) have revealed more than 80 genetic loci associated with COPD, leading to the identification of multiple COPD GWAS genes. However, the biological relationships between the identified COPD susceptibility genes are largely unknown. Genes associated with a complex disease are often in close network proximity, i.e. their protein products often interact directly with each other and/or similar proteins. In this study, we use affinity purification mass spectrometry (AP-MS) to identify protein interactions with HHIP, a well-established COPD GWAS gene which is part of the sonic hedgehog pathway, in two disease-relevant lung cell lines (IMR90 and 16HBE). To better understand the network neighborhood of HHIP, its proximity to the protein products of other COPD GWAS genes, and its functional role in COPD pathogenesis, we create HUBRIS, a protein-protein interaction network compiled from 8 publicly available databases. We identified both common and cell type-specific protein-protein interactors of HHIP. We find that our newly identified interactions shorten the network distance between HHIP and the protein products of several COPD GWAS genes, including DSP, MFAP2, TET2, and FBLN5. These new shorter paths include proteins that are encoded by genes involved in extracellular matrix and tissue organization. We found and validated interactions to proteins that provide new insights into COPD pathobiology, including CAVIN1 (IMR90) and TP53 (16HBE). The newly discovered HHIP interactions with CAVIN1 and TP53 implicate HHIP in response to oxidative stress.

Metabolomics describes the metabolic profile of an organism at a given time by the concentrations of its constituent metabolites. When studied over time, metabolite concentrations can help understand the dynamical evolution of a biological process. However, metabolites are involved into sequences of chemical reactions, called metabolic pathways, related to a given biological function. Accounting for these pathways into statistical methods for metabolomic data is thus a relevant way to directly express results in terms of biological functions and to increase their interpretability.

We propose a new method, phoenics, to perform differential analysis for longitudinal metabolomic data at the pathway level. In short, phoenics proceeds in two steps: First, the matrix of metabolite quantifications is transformed by a dimension reduction approach accounting for pathway information. Then, a mixed linear model is fitted on the transformed data.

This method was applied to semi-synthetic NMR data and two real NMR datasets assessing the effects of antibiotics and irritable bowel syndrome on feces. Results showed that phoenics properly controls the Type I error rate and has a better ability to detect differential metabolic pathways and to extract new impacted biological functions than alternative methods. The method is implemented in the R package phoenics available on CRAN.

Impaired differentiation is a hallmark of myeloid malignancies1,2. Therapies that enable cells to circumvent the differentiation block, such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), are by and large curative in acute promyelocytic leukaemia3, but whether 'differentiation therapy' is a generalizable therapeutic approach for acute myeloid leukaemia (AML) and beyond remains incompletely understood. Here we demonstrate that simultaneous inhibition of the histone demethylase LSD1 (LSD1i) and the WNT pathway antagonist GSK3 kinase4 (GSK3i) robustly promotes therapeutic differentiation of established AML cell lines and primary human AML cells, as well as reducing tumour burden and significantly extending survival in a patient-derived xenograft mouse model. Mechanistically, this combination promotes differentiation by activating genes in the type I interferon pathway via inducing expression of transcription factors such as IRF7 (LSD1i) and the co-activator β-catenin (GSK3i), and their selective co-occupancy at targets such as STAT1, which is necessary for combination-induced differentiation. Combination treatment also suppresses the canonical, pro-oncogenic WNT pathway and cell cycle genes. Analysis of datasets from patients with AML suggests a correlation between the combination-induced transcription signature and better prognosis, highlighting clinical potential of this strategy. Collectively, this combination strategy rewires transcriptional programs to suppress stemness and to promote differentiation, which may have important therapeutic implications for AML and WNT-driven cancers beyond AML.

Organismal aging has been associated with diverse metabolic and functional changes across tissues. Within the immune system, key features of physiological hematopoietic cell aging include increased fat deposition in the bone marrow, impaired hematopoietic stem and progenitor cell (HSPC) function, and a propensity towards myeloid differentiation. This shift in lineage bias can lead to pre-malignant bone marrow conditions such as clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenias of undetermined significance (CCUS), frequently setting the stage for subsequent development of age-related cancers in myeloid or lymphoid lineages. Human aging has also been associated with diverse lipid alterations across tissues, such as decreased phospholipid membrane fluidity that arises as a result of increased saturated fatty acid (FA) accumulation and a decay in n-3 polyunsaturated fatty acid (PUFA) species by the age of 80 years, however the extent to which impaired FA metabolism contributes to hematopoietic aging is less clear. Here, comprehensive multi-omics analyses uncovered a role for a key PUFA biosynthesis gene, ELOVL2, in mouse and human immune cell aging. Whole transcriptome RNA-sequencing studies and complementary flow cytometric analyses of bone marrow from aged Elovl2 mutant (enzyme-deficient) mice compared with age-matched controls revealed global downregulation in lymphoid cell markers and expression of genes involved specifically in B cell development. These studies unveiled CD79B, a vital molecular regulator of lymphoid progenitor development from the pro-B to pre-B cell stage, as a putative surface biomarker whose loss is associated with accelerated immune aging. The lipidome of mutant versus wild-type mice also displayed significant changes in the biophysical properties of cellular membranes. To investigate the relevance of these finding to human bone marrow aging, analyses of a single cell RNA-seq dataset of human HSPCs across the spectrum of human development and aging uncovered a rare subpopulation (< 7%) of CD34+ HSPCs that expresses ELOVL2 in healthy adult bone marrow. This HSPC subset, along with CD79B-expressing lymphoid-committed cells, were almost completely absent in CD34+ cells isolated from elderly bone marrow samples. Together, these findings uncover new roles for lipid metabolism enzymes in the molecular regulation of cellular aging and immune cell function in mouse and human hematopoiesis. In addition, because systemic loss of ELOVL2 enzymatic activity resulted in downregulation of B cell genes that are also associated with lymphoproliferative neoplasms, this study sheds light on an intriguing metabolic pathway that could be leveraged in future studies as a novel therapeutic modality to target blood cancers or other age-related conditions involving the B cell lineage.

Monocytes differentiate into macrophages (Mφs) to facilitate lung metastasis, but the monocyte-to-Mφ transition during this process is not well understood. To investigate, we performed bulk RNA sequencing on Mφs isolated from the lungs of mice bearing Lewis lung carcinoma tumors and from naive lungs. Our results showed impaired differentiation of monocytes into major histocompatibility complex (MHC) class II+ Mφs, with an upregulation of PGE2-inducible genes, including Arg1, in tumor-associated Mφs (TAMs). In vitro experiments confirmed that prostaglandin E2 (PGE2) inhibits the differentiation of MHC class II+ Mφs while promoting Arg1+ Mφs via the E prostanoid 2 (EP2) receptor, accompanied by DNA methylation. Whole-genome bisulfite sequencing revealed that PGE2-EP2 signaling drives the hypermethylation and downregulation of gene sets related to myeloid cells in non-neoplastic tissues. Our study highlights PGE2-EP2-driven DNA methylation in the monocyte-to-TAM transition, suggesting potential therapeutic avenues for lung metastasis.

Complement component C3 mediates pathology in CNS neurodegenerative diseases. Here we use scRNAseq of sorted C3-reporter positive cells from mouse brain and optic nerve to characterize C3 producing glia in experimental autoimmune encephalomyelitis (EAE), a model in which peripheral immune cells infiltrate the CNS, causing reactive gliosis and neuro-axonal pathology. We find that C3 expression in the early inflammatory stage of EAE defines disease-associated glial subtypes characterized by increased expression of genes associated with mTOR activation and cell metabolism. This pro-inflammatory subtype is abrogated with genetic C3 depletion, a finding confirmed with proteomic analyses. In addition, early optic nerve axonal injury and retinal ganglion cell oxidative stress, but not loss of post-synaptic density protein 95, are ameliorated by selective deletion of C3 in myeloid cells. These data suggest that in addition to C3b opsonization of post synaptic proteins leading to neuronal demise, C3 activation is a contributor to reactive glia in the optic nerve.

Although pluripotent stem cell (PSC) properties, such as differentiation and infinite proliferation, have been well documented within the frameworks of transcription factor networks, epigenomes, and signal transduction, they remain unclear and fragmented. Directing attention toward translational regulation as a bridge between these events can yield additional insights into previously unexplained mechanisms. Our functional CRISPR interference screen-based approach revealed that EIF3D, a translation initiation factor, is crucial for maintaining primed pluripotency. Loss of EIF3D disrupted the balance of pluripotency-associated signaling pathways, thereby compromising primed pluripotency. Moreover, EIF3D ensured robust proliferation by controlling the translation of various p53 regulators, which maintain low p53 activity in the undifferentiated state. In this way, EIF3D-mediated translation contributes to tuning the homeostasis of the primed pluripotency networks, ensuring the maintenance of an undifferentiated state with high proliferative potential. This study provides further insights into the translation network in maintaining pluripotency.

Translocation renal cell carcinoma (tRCC) presents a significant clinical challenge due to its aggressiveness and limited treatment options. It is primarily driven by fusion oncoproteins (FOs), yet their role in oncogenesis is not fully understood. Here, we investigate TFE3 fusions in tRCC, focusing on NONO::TFE3 and SFPQ::TFE3. We demonstrate that TFE3 FOs form liquid-like condensates with increased transcriptional activity, localizing to TFE3 target genes and promoting cell proliferation and migration. The coiled-coil domains (CCDs) of NONO and SFPQ are essential for condensate formation, prolonging TFE3 FOs' chromatin binding time and enhancing transcription. Compared with wild-type TFE3, TFE3 FOs bind to new chromatin regions, alter chromatin accessibility, and form new enhancers and super-enhancers at pro-growth gene loci. Disruption of condensate formation via CCD modification abolishes these genome-wide changes. Altogether, our integrated analyses underscore the critical functions of TFE3 FO condensates in driving tumor cell growth, providing key insights for future therapeutic strategies.

Sequencing of neuroblastoma (NB) tumors has revealed genetic alterations in genes involved in DNA damage response (DDR) pathways. However, roles for specific alterations of DDR genes in pediatric solid tumors remain poorly understood. To address this, mutations in the DDR pathway including Brca2, Atm, and Palb2 were incorporated into an established zebrafish MYCN transgenic model (Tg(dbh:EGFP-MYCN)). These mutations enhance NB formation and metastasis and result in upregulation of cell-cycle checkpoint and DNA damage repair signatures, revealing molecular vulnerabilities in DDR-deficient NB. DDR gene knockdown in zebrafish and human NB cells increases sensitivity to the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib, and this effect is enhanced by inhibition of the ataxia telangiectasia and rad3-related (ATR) kinase. This work provides in vivo evidence demonstrating that alterations in certain DDR-pathway genes promote aggressive NB and supports combination PARP + ATR inhibitor therapy for NB patients with tumors harboring specific genetic alterations in DDR.

Epithelia are tightly connected cellular sheets, that shield our body from the external environment. They are continuously maintained by a pooled population of undifferentiated cells through differentiation. However, the maintenance mechanisms remain incompletely understood due to the difficulty of experimentally observing the differentiation process. To address this issue, we developed a culture method for long-term expansion of primary mammary basal cells with a set of compounds, that includes undifferentiated cells. An effective differentiation method regarding Claudin expression was also developed by simply removing compounds. To verify this differentiation-switching technique, we obtained microarray data comparing each differentiation state. Subsequent cellular analysis confirmed key transcription factors in each state: (1) EGR1 in undifferentiated basal cells is important for suppressing Claudin expression through maintaining the epithelial-mesenchymal transition (EMT) transcription factor TWIST1, (2) ELF3 in differentiated cells is important for actin organization and subsequent Claudin localization at the cell-cell border, that corresponds to the amount of GRHL3, an actin organizer. Their relevance was also observed in tissues and organoids. In summary, we present an effective tool for verifying molecular mechanisms that determine Claudin status in normal basal cell differentiation, that would be beneficial in epithelial cell biology, cancer biology, physiology, and regeneration research.

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting articular joints and skeletal muscle. To assess the role of cytokines upon muscle strength in RA, we developed an in vitro tissue-engineered human skeletal muscle model (myobundle). Myobundles were generated using primary skeletal muscle cells from the vastus lateralis muscle of RA patients and age-matched healthy controls. RA myobundles were more sensitive to 5 ng/mL IFN-γ, exhibiting reduced contractile force and altered contraction kinetics. Addition of IL-6 with or without IFN-γ led to a small but significant increase in striated fibers. Gene sets involved in the response to hypoxia, MTOR1 signaling, and the unfolded protein response were enriched in IFN-γ-treated RA myobundles, but not IFN-γ-treated controls. Tofacitinib increased contractile force, myosin heavy chain, and PIM1 protein levels in RA myobundles treated with IFN-γ. Thus, in RA muscle, low levels of IFN-γ selectively increase gene pathways that reduce contractile force.

Colorectal carcinoma (CRC) exhibits metastatic organotropism, primarily targeting liver, lung, and rarely the brain. Here, we study chromosomal imbalances (CIs) in cohorts of primary CRCs and metastases. Brain metastases show the highest burden of CIs, including aneuploidies and focal CIs, with enrichment of +12p encoding KRAS. Compared to liver and lung metastases, brain metastases present with increased co-occurrence of KRAS mutation and amplification. CRCs with concurrent KRAS mutation and amplification display significant metabolic reprogramming with upregulation of glycolysis, alongside upregulation of cell cycle pathways, including copy number gains of MDM2 and CDK4. Evolutionary modeling suggests early acquisition of many organotropic CIs enriched in both liver and brain metastases, while brain-enriched CIs preferentially emerge later. Collectively, this study supports a model where cytogenetic events in CRCs favor site-specific metastatic colonization. These site-enriched CI patterns may serve as biomarkers for metastatic potential in precision oncology.

We previously discovered that HOXC6 was the most significantly upregulated gene in right-sided colon cancer compared to left-sided colon cancer according to our previous study; however, the role of HOXC6 in microsatellite instability-high (MSI-H) tumors remains poorly understood. Here, multiple public datasets, and in-house cohorts were used to analyze the differential expression and prognostic role of HOXC6 in colorectal cancer (CRC). Immunohistochemistry and immunofluorescence were performed to evaluate the correlation between HOXC6 expression and M2 macrophage infiltration. CCK8 and Transwell assays were used to evaluate the proliferation and migration of tumor cells in vitro. BALB/c nude mice were utilized to construct a humanized immune system model to evaluate the efficacy of ruxolitinib in vivo. We found that HOXC6 was overexpressed in MSI-H CRC and associated with a poor prognosis. Upregulation of CCL2 by HOXC6 increased M2 macrophage infiltration. IL6 secreted by M2 macrophages induced the epithelial-mesenchymal transition of tumor cells by upregulating HOXC6. M2 macrophages promoted effector T cell exhaustion by downregulating 4-1BB. Thus, inhibition of the IL6/JAK pathway in M2 macrophages restored 4-1BB expression and T-cell cytotoxicity offering a promising therapeutic target for the treatment of HOXC6-overexpressing MSI-H CRC.

Lung cancer is the leading cause of cancer-related deaths worldwide. Existing therapeutic options have limited efficacy, particularly for lung squamous cell carcinoma (LUSC), underscoring the critical need for the identification of new therapeutic targets. We previously demonstrated that the Transmembrane Serine Protease TMPRSS11B promotes transformation of human bronchial epithelial cells and enhances lactate export from LUSC cells. To determine the impact of TMPRSS11B activity on the host immune system and the tumor microenvironment (TME), we evaluated the effect of Tmprss11b depletion in a syngeneic mouse model. Tmprss11b depletion significantly reduced tumor burden in immunocompetent mice and triggered an infiltration of immune cells. RNA FISH analysis and spatial transcriptomics in the autochthonous Rosa26-Sox2-Ires-Gfp LSL/LSL ; Nkx2-1 fl/fl ; Lkb 1 fl/fl (SNL) model revealed an enrichment of Tmprss11b expression in LUSC tumors, specifically in Krt13 + hillock-like cells. Ultra-pH sensitive nanoparticle imaging and metabolite analysis identified regions of acidification, elevated lactate, and enrichment of M2-like macrophages in LUSC tumors. These results demonstrate that TMPRSS11B promotes an acidified and immunosuppressive TME and nominate this enzyme as a therapeutic target in LUSC.

Bifidobacteria represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest in bifidobacteria as a live biotic therapy, our understanding of colonization, host-microbe interactions, and the health-promoting effects of bifidobacteria is limited. To address these major knowledge gaps, we used a large-scale genetic approach to create a mutant fitness compendium in Bifidobacterium breve. First, we generated a high-density randomly barcoded transposon insertion pool and used it to determine fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. Second, to enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1,462 genes. We leveraged these tools to reveal community- and diet-specific requirements for colonization and to connect the production of immunomodulatory molecules to growth benefits. These resources will catalyze future investigations of this important beneficial microbe.