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PubMed2026

Zinc-dependent turnover of ZIP3 transporter mRNA by trypanosome ZNK1.

Like other cells, parasitic and other trypanosomatids sense Zn2+ and regulate Zn2+ transport, but the mechanisms involved remained unknown. Here, we identify a trypanosome RNA-binding protein that specifically eliminates ZIP3 transporter mRNA in Zn2+-replete conditions. We first demonstrate that Trypanosoma brucei ZIP3 mRNA abundance is subject to 3'-untranslated region (3'-UTR) and Zn2+-dependent negative control. A genome-wide RNA interference library screen, using a reporter associated with the ZIP3 3'-UTR, identifies Tb927.11.9510 as a candidate Zn2+-sensor, and we name this protein Zinc Nuclear Knuckles 1 (ZNK1) since it localizes to the nucleus and contains several Zn2+-knuckle motifs. ZNK1 is conserved among trypanosomatids, and a PIN domain suggests a ribonuclease-based mechanism. We use Cas9-editing to knockout ZNK1 and observe specific accumulation of ZIP3 transcripts, and increased intracellular Zn2+, in znk1 null cells. We validate ZNK1 as a ZIP3 3'-UTR-dependent negative regulator and identify a GU-repeat motif in the ZIP3 3'-UTR that is predictive of ZNK1-based negative control. In conclusion, ZNK1 eliminates ZIP3 transporter mRNA in a Zn2+-dependent manner. We suggest that trypanosomatid ZNK1 is a highly selective zinc finger nuclease that binds GU-repeat motifs within ZIP3 3'-UTRs and degrades Zn2+ transporter mRNA only when the tandem sensor knuckle modules are coordinated with Zn2+.

Trypanosoma brucei brucei↗
PubMed2026

Genetic basis of escape-related locomotor performance in a wild-introgressed sheep population.

Rapid running and jumping are core components of escape responses in prey animals and provide measurable traits for studying locomotor performance in large mammals. The genetic basis of these escape-related locomotor traits remains poorly understood in large mammals, partly because repeated, standardized phenotyping under field conditions is challenging. Here, we leveraged a sheep population carrying argali-introgressed genetic components to map genetic variations associated with running speed and jumping height. Through controlled field experiments, automated high-resolution phenotyping, whole-genome analysis, and gene-edited mouse models, we identified two loci associated with escape-related locomotor traits: one in ABCC4 (Chr10:71,849,347; p = 5.03 × 10-7) linked to maximum running speed and another in GRID2 (Chr6:32,120,477; p = 1.30 × 10-9) associated with jumping height. Functional assays in knockout mice reveal that disruption of Grid2 reduces jumping ability, whereas Abcc4 knockout and knockdown increase running speed through enhanced heart contractility under stress. These results elucidated the genetic bases of wild-derived variations in affecting locomotor performance.

Animals↗
PubMed2026

A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.

Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.

Animals↗
PubMed2026

Efficient scarless gene editing in Pichia pastoris via survival stress-based intramolecular homologous recombination.

To overcome low efficiency and/or genomic instability induced by DNA cleavage in current genome-editing approaches, a novel pop-in/pop-out-based editing system was developed for Pichia pastoris. An ingenious arrangement of components leads to a more efficient screening by permitting the only type of DNA recombination under defined pressure conditions, in terms of the overall efficiency of gene editing, the system virtually depends on the integration efficiency mediated by single-crossover recombination. It does not rely on exogenous recombinases or programmable nucleases such as Cas9, thereby avoiding nuclease induced double strand breaks and associated off target mutations or chromatin fatigue. This strategy preserves high editing efficiency with no modification to the host's inherent genetic properties. Relative to site-specific recombination methods, its dual MazF counterselection enables seamless editing, avoiding scar sequence-induced genomic instability. In this study, nearly 100% knockout efficiency and over 86.67% integration efficiency were achieved in the described experimental cases with this system, which provides a new gene-editing tool for synthetic biology in Pichia pastoris.

Efficient scarless editing↗
PubMed2026

Anti-psychotic drugs act synergistically in combination with antifungal drugs to inhibit drug-resistant Cryptococcus neoformans and Candida albicans.

UNLABELLED: Systemic fungal infections cause an estimated 3.8 million deaths annually, approximately 10% of which are caused by drug-resistant infections. With only five classes of antifungal drugs, treatment options are limited. Here, we explore synergistic drug combinations-when the efficacy of two drugs combined is greater than expected based on the sum of each individual drug's efficacy-to improve treatment of drug-resistant Cryptococcus neoformans and Candida albicans. Chlorpromazine acts synergistically with both amphotericin B and fluconazole against multiple fungal species, including azole-resistant C. neoformans and C. albicans. We then performed a genome-wide knockout mutant screen and found that ESCRT pathway mutants are resistant to chlorpromazine, while knockout mutants of genes involved in fatty acid biosynthesis are sensitive. Based on these data, we investigated sterol and fatty acid composition in chlorpromazine-treated cells and found only minor increases in sterol precursors, but a substantial increase in lipid droplet size and decreased lipid droplet numbers. This lipid droplet formation potentially sequesters lipid bioavailability and response to membrane stress. Together, these data suggest that chlorpromazine and its analogs are potentially promising treatments for systemic fungal infections that act via lipid homeostasis and stress response. IMPORTANCE: Fungal infections are a large and expensive health burden with high mortality rates. People with compromised immune systems from cancer, solid organ transplant, HIV infection, and other conditions are particularly affected. Systemic fungal infections are difficult to treat because there are few available drugs and treatment periods last months or years. Long treatment times increase the risk of treatment failure and can contribute to the rise of resistance. We identified an additional class of drugs, chlorpromazine and other phenothiazine drugs, that amplify the activity of existing antifungal drugs amphotericin B (AmB) and fluconazole (FLZ). AmB and FLZ act by targeting ergosterol, the fungal equivalent of cholesterol, which is required for a functional plasma membrane. Chlorpromazine increases the formation of lipid drops, which sequester lipids such as ergosterol. When chlorpromazine is combined with AmB, the fungal cell cannot respond to the plasma membrane damage caused by AmB, inhibiting the fungal cells. This work identifies new target processes and drugs that could treat deadly fungal infections.

antifungal resistance↗
PubMed2025

Ultra-high field strength electroporation enables efficient DNA transformation and genome editing in nontuberculous mycobacteria.

Efficient DNA delivery is essential for genetic manipulation of mycobacteria and for dissecting their physiology, pathogenesis, and drug resistance. Although electroporation enables transformation efficiencies exceeding 10⁵ CFU per µg DNA in Mycobacterium smegmatis and Mycobacterium tuberculosis, it remains highly inefficient in many nontuberculous mycobacteria (NTM), including Mycobacterium abscessus. Here, we discovered that NTM such as M. abscessus exhibit exceptional tolerance to ultra-high electric field strengths and that hypertonic preconditioning partially protects cells from electroporation-induced damage. Using ultra-high electric field strength (3 kV/mm) electroporation, we achieved dramatic improvements in plasmid transformation efficiency-up to 106-fold in M. abscessus, 83-fold in Mycobacterium marinum, and 37-fold in Mycobacterium kansasii-compared to standard conditions (1.25  kV/mm). Transformation efficiency was further influenced by the choice of selectable marker. Ultra-high field strength electroporation also markedly enhanced allelic exchange in M. abscessus expressing Che9c RecET recombinases, increasing the recovery of gene deletion mutants by over 1,000-fold relative to conventional electroporation. In parallel, oligonucleotide-mediated recombineering for targeted point mutations produced nearly 10,000-fold more mutants under ultra-high field conditions. Together, these findings establish ultra-high field electroporation as a robust, broadly applicable platform for genetic engineering of NTMs. This method substantially enhances transformation efficiency and enables construction of advanced genetic tools-including expression libraries and CRISPRi knockdown libraries-in species that have historically resisted genetic manipulation.IMPORTANCEInfections caused by nontuberculous mycobacteria (NTM), including Mycobacterium abscessus, are increasing globally, yet genetic manipulation of these pathogens remains technically challenging due to inefficient DNA delivery and low gene editing success. The ultra-high electric field strength electroporation strategy described here overcomes these barriers, enabling dramatic improvements in both transformation and genome editing efficiency. This advance paves the way for high-throughput functional genomics in NTMs, including the construction of genome-wide knockout, CRISPRi knockdown, and expression libraries. Broad adoption of this approach will accelerate discovery of genetic determinants of virulence and drug resistance, facilitating the development of antimicrobials and vaccines.

Electroporation↗
PubMed2025

Tdrd15 is dispensable for male fertility and spermatogenesis in the golden hamster.

Tudor domain-containing proteins (TDRDs) constitute an evolutionarily conserved protein family and are critical for germline development and piRNA pathway regulation, with established roles in male fertility. While multiple TDRD family members have been functionally linked to spermatogenic impairment, the precise biological role of TDRD15 remains to be elucidated. We used CRISPR/Cas9-mediated gene editing to generate Tdrd15 knockout (KO) golden hamsters (Mesocricetus auratus), a model necessitated by the absence of a functional Tdrd15 ortholog in the mouse genome, to investigate its function in male reproduction. Phylogenetic analysis demonstrated that TDRD15 is strongly conserved among eutherian mammals, with testis-restricted expression patterns in hamsters. Despite the successful induction of frameshift mutations and significant transcriptional knockdown, Tdrd15 KO males maintained normal fertility parameters, including unaltered testicular architecture, spermatogenic progression (confirmed by periodic acidic-Schiff (PAS) staining and immunohistochemistry), and sperm quality metrics determined using a computer-assisted analysis. Quantitative polymerase chain reaction (qPCR) revealed compensatory overexpression of paralogous Tdrd genes in KO testes, implying functional redundancy within this protein family. This study provides the first experimental evidence that TDRD15 is dispensable for male fertility in golden hamsters under physiological conditions, thereby challenging the prevailing assumptions of its obligatory function in spermiogenesis. Altogether, these findings support a more targeted allocation of research efforts within the field of male reproductive biology.

Animals↗
PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗
PubMed2026

A novel G13-RAPGEF2-RAP1 signaling pathway critical for platelet adhesion and aggregation.

Hemostasis and thrombosis are strongly dependent on the unique ability of platelets to rapidly activate integrin receptors and to firmly adhere to sites of injury under shear stress conditions. Central to integrin activation is the small GTPase RAP1, which itself is activated by guanine nucleotide exchange factors (GEFs). CalDAG-GEFI (RASGRP2) is the highest expressed and functionally dominant platelet RAP-GEF. However, a genome-wide association study also suggested a significant role for RAPGEF2 (PDZ-GEFI), a low-expressed RAP-GEF, in human platelet aggregation. Here, we used mice deficient in RAPGEF2 (megakaryocyte-specific, Rapgef2mKO), CalDAG-GEFI (Caldaggef1-/-), or both RAPGEF2 and CalDAG-GEFI (DKO) to characterize the contribution of RAPGEF2 signaling to platelet function, hemostasis, and thrombosis. RAPGEF2 protein was detected in murine and human platelets. Compared with control or Caldaggef1-/- platelets, both RAP1 activation and integrin αIIbβ3-mediated aggregation were significantly diminished in DKO platelets. When compared with controls, Rapgef2mKO platelets exhibited reduced integrin activation, a more reversible aggregation response, and impaired adhesion under conditions of shear stress ex vivo and in vivo. Mechanistic studies strongly suggest that RAPGEF2 operates downstream of receptors coupled to the heterotrimeric G protein G13 (GNA13), such as αIIbβ3 and the thromboxane receptor. Together, our studies provide genetic evidence that RAPGEF2 in platelets operates downstream of G13 as an important regulator of RAP1 signaling and integrin activation, especially under conditions of elevated shear stress. These findings markedly improve our understanding of G protein signaling and integrin function in platelets, with potential implications for the development of improved platelet-targeted therapies for cardiovascular disease.

Animals↗
PubMed2026

CD109 exhibits a dynamic expression pattern in coronary endothelium and endocardial-derived valve mesenchyme during heart development with preserved morphogenesis following endothelial-specific deletion.

BACKGROUND: CD109 encodes a GPI-linked glycoprotein that acts as a signaling modulator in the TGF-β pathway. CD109 has emerged in several genome-wide association studies as linked to coronary artery disease, myocardial infarction, and angina pectoris. Heterozygous loss-of-function mutations in CD109 have also been reported in patients with congenital heart defects, suggesting potential developmental relevance, though CD109 has never been investigated in the context of cardiovascular development. We previously identified Cd109 upregulation in murine atrioventricular valves undergoing myxomatous degeneration following a reduction of epicardial-derived cells. Here, we characterize Cd109 expression in the murine cardiovascular system and assess its function during development using in vitro and in vivo approaches. RESULTS: We found that Cd109 is strongly expressed in the endothelium of the coronary vasculature and in endocardial-derived subpopulations in the atrioventricular valves. This expression persists through key stages in cardiovascular development. Western blotting and immunostaining confirm endothelial expression in heart and lung tissues. siRNA-mediated knockdown of CD109 in primary human endothelial cells led to dysregulation of vascular development pathways and decreased tube formation capacity. We generated endothelial-specific Cd109 knockout mice, eliminating Cd109 expression from heart and lung tissues without overt consequences for atrioventricular valve or coronary vascular morphogenesis during heart development. CONCLUSION: CD109 exhibits a highly dynamic spatiotemporal expression pattern during cardiovascular development, with enriched expression in coronary endothelial cells and endocardial-derived subpopulations in the valves. Despite this striking developmental expression pattern, previously reported human genetic associations with cardiovascular diseases, and endothelial-associated phenotypes following siRNA-mediated CD109 knockdown in a primary human endothelial cell line, endothelial/endocardial-lineage deletion of Cd109 did not produce overt abnormalities in atrioventricular valve or coronary vascular morphogenesis during embryonic development. Collectively, these findings identify CD109 as a useful marker of coronary endothelial and endocardial-derived valve cell populations and suggest that CD109 may function in a context-dependent or modulatory manner rather than as an essential regulator of cardiovascular morphogenesis under normal developmental conditions.

CD109↗
PubMed2025

Deubiquitinase-dependent transcriptional silencing controls inflammation.

Transcriptional control is crucial for the regulation of inflammation. While it is well-established that inducible transcriptional repressors are synthesized de novo through signal-dependent transcriptional upregulation, it remains unclear whether post-translational modification mechanisms, such as deubiquitination, also contribute to this process. We previously identified developmentally silenced sine oculis (SIX) transcription factors that are reactivated to control inflammatory gene transcription in differentiated immune cells under chronic microbial infections. However, the molecular mechanisms by which this transcriptional silencing process is regulated remain unclear. Here, we report that USP2, a deubiquitinase localized in the nucleus and induced by inflammatory signals, stabilizes SIX proteins through deubiquitination under inflammatory conditions. Consequently, the USP2-SIX complex acts in concert to control NF-κB-mediated inflammatory gene transcription by directly targeting gene promoters. Supporting this mechanism, Usp2-/- mice exhibit higher mortality during H1N1 infections, which phenocopies Six1-/- mice, attributed to elevated levels of life-threatening inflammatory mediators and exacerbated pathology. This study establishes a deubiquitinase-dependent transcriptional control of the inflammatory response to prevent immunopathology, offering new therapeutic avenues for combating infectious diseases.

Animals↗
PubMed2026

RNA Binding Protein PCBP1 Functions in the Endothelial-to-Hematopoietic Transition During Hematopoietic Stem Cell Formation.

Two paralogous RNA-binding proteins, PCBP1 and PCBP2, are individually essential for mouse development. Pcbp2-null embryos lose viability at midgestation, whereas Pcbp1-null embryos have an earlier, peri-implantation lethality. Inactivation of each gene alone in the differentiating erythroid lineage failed to impact embryonic viability, whereas combined Pcbp1/Pcbp2 inactivation resulted in repression of erythroid/hematopoietic gene expression midgestation, decreased blood cell formation, and fetal demise. Here, we assess the impacts of individual and combined conditional inactivation of Pcbp1 and Pcbp2 in endothelial cells on embryonic hematopoietic stem and progenitor cell (HSPC) formation. Inactivation of Pcbp1 in endothelial cells is embryonic lethal and reduces the number of HSPCs, whereas loss of Pcbp2 has no impact. Combined deletion of Pcbp1 and Pcbp2 in endothelial cells results in a more severe phenotype than loss of Pcbp1 alone. These results demonstrate that although Pcbp1 is a major regulator of HSPC formation, the two genes have nonredundant roles in endothelial cells.

Animals↗
PubMed2026

An inflammatory bowel disease-linked lncRNA suppresses transcription factor T-BET expression in T cells to limit intestinal inflammation.

Among the tens of thousands of annotated long noncoding RNAs (lncRNAs) in the human genome, only a small fraction have been functionally characterized. Here, we show that a well-established inflammatory bowel disease (IBD) risk locus encoded a conserved lncRNA, lnc15 (2310015A10Rik/ENSMUSG00000097729), whose structure was destabilized by risk-associated variants, leading to its degradation. Deletion of lnc15 in mice resulted in molecular features of inflammation under steady-state conditions and conferred heightened susceptibility to experimental colitis. Lnc15 was abundantly expressed in T cells, with highest expression in regulatory T (Treg) cells. Mechanistically, lnc15 suppressed the transcription factor T-BET by recruiting the CCR4-NOT RNA degradation complex to Tbx21 mRNA. Our study identifies that lnc15 simultaneously enhances Treg cell suppressive function and impairs conventional T cell pathogenicity in the context of intestinal inflammation. Collectively, these findings identify lnc15 as a functional lncRNA that links noncoding genetic variation to immune regulation and prevention of mucosal inflammation. VIDEO ABSTRACT.

RNA, Long Noncoding↗
PubMed2026

Epidermal NAD+ deficiency induces IL-36-mediated skin inflammation and acanthosis.

Nicotinamide adenine dinucleotide (NAD+) is essential for cellular metabolism, DNA repair, and stress responses. NAD+ is synthesized from nicotinamide, nicotinic acid (collectively termed niacin), and tryptophan. In humans, deficiencies in these nutrients result in pellagra, marked by dermatitis, diarrhea, and dementia. The dermatitis associated with pellagra typically manifests as photodermatosis in sun-exposed areas. This study examined the effects of NAD+ deficiency on skin homeostasis using epidermis-specific Nampt-conditional KO (Nampt-cKO) mice. These mice displayed substantial NAD+ depletion, reduced poly(ADP-ribose) polymerase (PARP) activity, and increased DNA damage. Consequently, Nampt-cKO mice developed spontaneous skin inflammation and epidermal hyperplasia. RNA-seq and IHC analyses demonstrated increased IL-36 cytokine expression, suggesting that DNA repair-related genomic stress triggers keratinocyte-driven IL-36 production, which promotes inflammation. Furthermore, reduced COL17A1 expression and elevated thymic stromal lymphopoietin (TSLP) levels were observed. NAD+ repletion by transdermal supplementation of nicotinamide mononucleotide (NMN) suppressed the rise of IL-36 levels and skin inflammation. These findings underscore the importance of Nampt-mediated NAD+ metabolism for epidermal stability and indicate that NAD+ depletion may contribute to IL-36-mediated skin inflammation, offering insights for therapeutic strategies in inflammatory skin disorders.

Animals↗
PubMed2026

Tropomyosin 1 Promotes Platelet Adhesion and Clot Contraction Separate from Its Roles in Developmental Hematopoiesis.

Genome-wide association studies (GWAS) link the Tropomyosin 1 (Tpm1) locus to quantitative blood trait variation, but related mechanisms are unclear. Tpm1 encodes an actin-binding protein that regulates actin filament diversity, cell adhesion, signaling, and actomyosin contractility. Murine Tpm1 deficiency enhances hemogenic endothelial cell (HEC) specification, but it was unclear if these effects extended to postnatal hematopoiesis. We used Cdh5Cre and VavCre models to conditionally knock out Tpm1 (Tpm1KO) in endothelial anor hematopoietic cells. Both models ablate Tpm1 in postnatal blood. Endothelial Tpm1KO increases HEC specification without altering hematopoietic progenitor cell production or adult blood counts, suggesting separate roles for Tpm1 in the embryonic and adult blood systems. Tpm1KO increases adult platelet lifespan and diminishes adhesion to fibronectin and fibrinogen. Chemical Tpm1 inhibition also reduces focal adhesion in murine and human platelets. Altered platelet morphology and reduced platelet spreading suggest perturbed actomyosin contractility underlies these findings. Platelet fibrin binding promotes blood clot contraction, which reduces occlusive thrombosis. Tpm1KO limits clot contraction and worsens vascular occlusion in ferric chloride-induced stroke models. In addition to offering a mechanistic explanation for why genetic variation at the TPM1 alters platelet traits in GWAS, our findings reveal novel roles for Tpm1 in clot contraction and thrombosis.

Animals↗
PubMed2025

IGF1R deficiency mitigates acute lung injury by promoting anti-inflammatory transcriptional profiles.

BACKGROUND: Acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and COVID-19 are characterized by hyperinflammation, commonly referred to as "cytokine storm". The insulin-like growth factor (IGF) pathway, particularly the type 1 receptor (IGF1R), plays a critical role in lung homeostasis and has been implicated in the pathogenesis of pulmonary inflammatory diseases. In mice, widespread Igf1r deficiency attenuates lung inflammation and alveolar damage in bleomycin (BLM)-induced ALI. METHODS: We analyzed single-cell RNA sequencing datasets from lung tissue of COVID-19 cases and control donors as well as mouse lungs to determine Igf1r and IGF family expression across pulmonary cell types. Furthermore, we conducted bulk RNA sequencing on lungs from Igf1r-deficient mice three days after BLM or saline instillation, followed by differential expression and functional enrichment analyses. Findings were further tested through protein detection, assessment of DNA damage and methylation in lung tissues, and functional assays using Igf1r-deficient primary mouse embryonic fibroblasts (MEFs). RESULTS: IGF1R was broadly expressed across multiple cell types in both human and mouse lungs under normal and pathological conditions. Other IGF family members showed cell-type-specific expression, which was modulated by lung injury. Transcriptomic profiling revealed differentially expressed genes between BLM-challenged and control mouse lungs, detecting biological processes and signaling pathways involved in ALI pathobiology. Igf1r deficiency in BLM-challenged mice reversed a large fraction of the transcriptional changes triggered by BLM, including "cytokine storm"-related gene expression. Functional enrichment analysis additionally revealed significant modulation of pathways related to DNA damage, metabolic reprogramming, mitochondrial homeostasis, and epigenetic regulation. In vitro, Igf1r-deficient MEFs exhibited decreased mitochondrial respiration and glycolysis, protection against BLM-induced nuclear damage and mitochondrial accumulation, and decreased histone H3 acetylation. Moreover, Igf1r-deficient mouse lungs displayed increased global DNA methylation following BLM challenge. CONCLUSIONS: IGF1R is a key modulator of the inflammatory and molecular response to ALI pathogenesis. IGF1R deficiency dampens the "cytokine storm", modifies transcriptional and epigenetic profiles and promotes protective cellular responses. These findings highlight IGF1R signaling as a potential therapeutic target in ARDS and related lung injuries.

Animals↗
PubMed2026

Dietary arginine drives codon-dependent MHC class I translation and improves immunity in colon tumorigenesis and respiratory viral infection.

Amino acid levels fluctuate across diverse pathological conditions. Whether such amino acid modulations directly shape pathophysiology by regulating host gene expression remains unknown. We found that extracellular arginine restriction, observed in cancer and infection, represses specific arginine tRNAs-directly suppressing translation of major histocompatibility complex I (MHC class I) and antigen presentation. Arginine regulation of MHC class I was codon-usage dependent, as synonymous codon mutations prevented MHC class I modulation. Dietary arginine restriction impaired anti-viral immunity against influenza and SARS-CoV-2 and increased colon tumorigenesis. Conversely, increasing arginine availability via dietary supplementation or myeloid-specific arginase 1 deletion enhanced MHC class I protein levels, suppressed colon tumorigenesis, and improved viral infection outcomes. These disease-modulating effects were abolished in β2-microglobulin (B2m)-deficient mice. Thus, dietary modulation of a single amino acid critically influences codon-biased translation and MHC class I-mediated immunity to respiratory viral infections and cancer, revealing an unexpected mechanism and disease hazard for arginine deficiency and highlighting potential for amino acid-based translation modulation therapy.

Animals↗
PubMed2017

Ischemia/Reperfusion Induces Interferon-Stimulated Gene Expression in Microglia.

Innate immune signaling is important in the pathophysiology of ischemia/reperfusion (stroke)-induced injury and recovery. Several lines of evidence support a central role for microglia in these processes. Recent work has identified Toll-like receptors (TLRs) and type I interferon (IFN) signaling in both ischemia/reperfusion-induced brain injury and ischemic preconditioning-mediated neuroprotection. To determine the effects of "ischemia/reperfusion-like" conditions on microglia, we performed genomic analyses on wild-type (WT) and TLR4-/- cultured microglia after sequential exposure to hypoxia/hypoglycemia and normoxia/normoglycemia (H/H-N/N). We observed increased expression of type 1 IFN-stimulated genes (ISGs) as the predominant transcriptomal feature of H/H-N/N-exposed WT, but not TLR4-/-, microglia. Microarray analysis on ex vivo sorted microglia from ipsilateral male mouse cortex after a transient in vivo ischemic pulse also demonstrated robust expression of ISGs. Type 1 IFNs, including the IFN-αs and IFN-β, activate the interferon-α/β receptor (IFNAR) complex. We confirmed both in vitro H/H-N/N- and in vivo ischemia/reperfusion-induced microglial ISG responses by quantitative real-time PCR and demonstrated that both were dependent on IFNAR1. We characterized the effects of hypoxia/hypoglycemia on phosphorylation of signal transducer and activator of transcription 1 (STAT1), release of type 1 IFNs, and surface expression of IFNAR1 in microglia. We demonstrated that IFN-β induces dose-dependent secretion of ISG chemokines in cultured microglia and robust ISG expression in microglia both in vitro and in vivo Finally, we demonstrated that the microglial ISG chemokine responses to TLR4 agonists were dependent on TLR4 and IFNAR1. Together, these data suggest novel ischemia/reperfusion-induced pathways for both TLR4-dependent and -independent, IFNAR1-dependent, type 1 IFN signaling in microglia.SIGNIFICANCE STATEMENT Stroke is the fifth leading cause of death in the United States and is a leading cause of serious long-term disability worldwide. Innate immune responses are critical in stroke pathophysiology, and microglia are key cellular effectors in the CNS response to ischemia/reperfusion. Using a transcriptional analysis approach, we identified a robust interferon (IFN)-stimulated gene response within microglia exposed to ischemia/reperfusion in both in vitro and in vivo experimental paradigms. Using a number of complementary techniques, we have demonstrated that these responses are dependent on innate immune signaling components including Toll-like receptor-4 and type I IFNs. We have also elucidated several novel ischemia/reperfusion-induced microglial signaling mechanisms.

Animals↗