Biology subjects

Ma, X.

Publications and source records attributed to Ma, X..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Unveiling Protein-DNA Interdependency: Harnessing Unified Multimodal Sequence Modeling, Understanding and Generation

The interdependent orchestration of proteins, coding DNA sequences (CDS), and adjacent regulatory DNA elements underpins biological functionality, where evolutionary constraints shape codon usage, transcriptional regulation, and structural stability. We present ProDMM, a multimodal framework that deciphers these systemic relationships through unified sequence modeling. Integrating a context-aware encoder with conditional generation, our architecture captures codependent patterns between protein motifs and cis-regulatory syntax. ProDMMs encoder demonstrates superior predictive capabilities in forecasting pathway-level phenotypes and protein expression profiles, outperforming conventional single-modality approaches. Its decoder generates synthetically optimized regulatory elements by resolving spatial dependencies between coding sequences and adjacent non-coding regions, enabling transcriptional fine-tuning unavailable to sequence-agnostic methods. By resolving how localized CDS-NCDS-protein interdependencies propagate to system-level phenotypes, ProDMM establishes a paradigm for holistic biological sequence engineering. This approach enables coordinated optimization of translation efficiency, enzymatic activity, and pathway flux, offering transformative potential for designing tailored biosynthesis systems and advancing sustainable bioproduction strategies.

bioinformatics↗

bioRxiv2025

scWGBS-GPT: A Foundation Model for Capturing Long-Range CpG Dependencies in Single-Cell Whole-Genome Bisulfite Sequencing to Enhance Epigenetic Analysis

Single-cell DNA methylomes are challenging to interpret because of sparse CpG coverage and the complexity of genome-wide sequences. We present scDNAm-GPT, a universal foundation model that uses context-aware CpG tokenization, a Mamba backbone, and cross-attention to capture both local and global DNA methylation patterns. Trained on over one million single cells from 35 human and mouse tissues, scDNAm-GPT enables accurate cell clustering, zero-shot prediction of CpG effects on gene expression, improved trajectory inference, and reference-free deconvolution of cell types from cell-free DNA. The model hierarchically learns regulatory features, and its attention maps highlight functionally relevant regions, demonstrating high biological interpretability. These results establish scDNAm-GPT as a scalable and generalizable framework for single-cell epigenomic analysis, offering new opportunities to dissect epigenetic regulation in health and disease. Code is available at GitHub (https://github.com/ChaoqiLiang/scDNAm-GPT).

bioinformatics↗

bioRxiv2025

Cloche/Npas4l is a pro-regenerative platelet factor during zebrafish heart regeneration

Zebrafish has full capacity of heart regeneration, but little is known about how blood cells, especially platelets, are involved in this regenerative process. Here we report that cloche/npas4l is a pro-regenerative platelet factor for heart regeneration. We found that haploinsufficiency of npas4l disrupted cardiomyocyte (CM) and endothelial cell (EC) proliferation and heart regeneration after injury. A single-cell transcriptomic atlas revealed that npas4l was dynamically expressed in platelets after heart injury and regulated robust interactions between platelets-CMs or -ECs. Decreasing platelets by either nitroreductase-mediated platelet depletion or platelet-deficient mutant mpl impaired CM and EC proliferation, and over-expression of npas4l in platelets sufficiently induced CM and EC proliferation in uninjured hearts, as well as rescued CM and EC proliferation defects in cloche mutants. Mechanistically, Npas4l positively regulated a panel of ligands expression including bmp6 in platelets to fine-tune CM proliferation and heart regeneration. Therefore, this work unveils a novel platelet Npas4l signaling and presents mechanisms on how platelets modulate CM/EC proliferation via ligand-receptor network during zebrafish heart regeneration.

cell biology↗

bioRxiv2025

Comparative molecular landscapes of immature neurons in the mammalian dentate gyrus across species reveal special features in humans

Immature dentate granule cells (imGCs) arising from adult hippocampal neurogenesis contribute to plasticity, learning and memory, but their evolutionary changes across species and specialized features in humans remain poorly understood. Here we performed machine learning-augmented analysis of published single-cell RNA-sequencing datasets and identified macaque imGCs with transcriptome-wide immature neuronal characteristics. Our cross-species comparisons among humans, monkeys, pigs, and mice showed few shared (such as DPYSL5), but mostly species-specific gene expression in imGCs that converged onto common biological processes regulating neuronal development. We further identified human-specific transcriptomic features of imGCs and demonstrated functional roles of human imGC-enriched expression of a family of proton-transporting vacuolar-type ATPase subtypes in development of imGCs derived from human pluripotent stem cells. Our study reveals divergent gene expression patterns but convergent biological processes in the molecular characteristics of imGCs across species, highlighting the importance of conducting independent molecular and functional analyses for adult neurogenesis in different species.

neuroscience↗

bioRxiv2025

Disruption of Mitochondrial Dynamics and Stasis Leads to Liver Injury and Tumorigenesis

Background & AimsMitochondrial dysfunction has been implicated in aging and various cancer development. As highly dynamic organelles, mitochondria constantly undergo fission, mediated by dynamin-related protein 1 (DRP1, gene name Dnm1l), and fusion, regulated by mitofusin 1 (MFN1), MFN2, and optic atrophy 1 (OPA1). However, whether and how dysregulation of mitochondria dynamics would be involved in liver pathogenesis and tumorigenesis is unknown. MethodsDnm1l Flox/Flox (Dnm1lF/F), Mfn1F/F and Mfn2F/F mice were crossed with albumin-Cre mice to generate liver-specific Dnm1l knockout (L-Dnm1l KO), L-Mfn1 KO, L-Mfn2 KO, L-Mfn1, Mfn2 double KO (DKO), and L-Mfn1, Mfn2, Dnm1l triple KO (TKO) mice. These mice were housed for various periods up to 18 months. Some mice also received hydrodynamic tail vein injections of a Sleeping Beauty transposon-transposase plasmid system with c-MYC and YAP. Blood and liver tissues were harvested for biochemical and histological analysis. ResultsL-Dnm1l KO mice had elevated serum alanine aminotransferase levels and increased hepatic fibrosis as early as two months of age. By 12 to 18 months, male L-Dnm1l KO mice developed spontaneous liver tumors, primarily hepatocellular adenomas. While female L-Dnm1l KO mice also developed liver tumors, their incidence was much lower. In contrast, neither L-Mfn1 KO nor L-Mfn2 KO mice had notable liver injury or tumorigenesis. However, a small portion of DKO mice developed tumors at 15-18 month-old. Increased DNA damage, senescence and compensatory proliferation were observed in L-Dnm1l KO mice but were less evident in L-Mfn1 KO, L-Mfn2 KO or DKO mice, indicating that mitochondrial fission is more important to maintain hepatocyte homeostasis and prevent liver tumorigenesis. Interestingly, further deletion of Mfn1 and Mfn2 in L-Dnm1l KO mice markedly abolished liver injury, fibrosis, and both spontaneous and oncogene-induced tumorigenesis. RNA sequencing and metabolomics analysis revealed significant activation of the cGAS-STING-interferon pathway and alterations in the tumor microenvironment pathways, alongside increased pyrimidine synthesis and metabolism in the livers of L-Dnm1l KO mice. Notably, the changes in gene expression and pyrimidine metabolism were considerably corrected in the TKO mice. ConclusionsMitochondrial dynamics and stability are essential for maintaining hepatic mitochondrial homeostasis and hepatocyte functions. Loss of hepatic DRP1 promotes liver tumorigenesis by increasing pyrimidine metabolism and activating the cGAS-STING-mediated innate immune response.

cell biology↗

bioRxiv2025

Cystinosin is involved in Na+/H+ Exchanger 3 trafficking in the proximal tubular cells: new insights in the renal Fanconi syndrome in cystinosis

Cystinosis is a systemic lysosomal storage disease resulting from a defective CTNS gene, leading to the accumulation of cystine in all organs. Despite the ubiquitous expression of cystinosin, the renal Fanconi syndrome (FS) is the first manifestation of cystinosis that presents early in life of the patients while other complications appear years later. Additionally, the cystine reduction therapy, cysteamine, does not prevent the FS. While the matter is still unresolved, it is apparent that specific function(s) of cystinosin in the proximal tubular cells (PTCs) beyond cystine transport explain the early tubular defects in cystinosis. Here, we report a novel interaction of cystinosin with the sodium/hydrogen (Na+/H+) exchanger proteins in the endosomes in both yeast and mammalian cells. One isoform of Na+/H+ exchanger, NHE3, is a major absorptive sodium transporter at the apical membrane of the proximal tubules. Cystinosin was found to play a significant role in NHE3 subcellular localization, trafficking, and resulting sodium uptake in PTCs. Interestingly, introduction of CTNS successfully rescued these defects in CTNS-deficient PTCs, whereas CTNS-LKG, the lysosomal and plasma membrane isoform of cystinosin, did not. NHE3 mislocalization was confirmed in Ctns-/- mice and cystinosis patient kidney. Interestingly, transplantation of wild-type hematopoietic stem and progenitor cells in Ctns-/- mice restored NHE3 expression at the brush border membrane. This study uncovers a new role of cystinosin in the trafficking of NHE3 in the PTCs that is evolutionary conserved, offering new insights in the pathogenesis of the renal FS in cystinosis and potential new therapeutic avenue for this pathology. SignificanceThis study reveals a new role of cystinosin in the trafficking of NHE3, the Na+/H+ exchanger in the proximal tubular cells (PTCs) in the kidney. NHE3 contributes to the majority of renal sodium absorption and is essential for maintaining the homeostasis of the PTCs. Thus, this study offers new molecular insights in the endosomal trafficking of NHE3 and provides a new mechanism that may explain the underlying pathogenesis and early onset of the renal Fanconi syndrome in cystinosis. Additionally, these findings provide potential new drug targets for this pathology and are also relevant to our ongoing stem cell gene therapy clinical trial for cystinosis, offering valuable information on the potential impact of hematopoietic stem cell transplantation on renal Fanconi syndrome.

molecular biology↗

bioRxiv2025

Glycosylphosphatidylinositol biosynthesis restricts coronavirus infection via the regulation of LY6E

Coronaviruses, including SARS-CoV-2, rely on host factors for their replication and pathogenesis, while hosts deploy defense mechanisms to counteract viral infections. Although numerous host proviral factors have been identified, the landscape of host restriction factors and their underlying mechanisms remain less explored. Here, we conducted genome-wide CRISPR knockout screens using three distinct coronaviruses--SARS-CoV-2, HCoV-OC43 (a common cold human virus from the genus Betacoronavirus) and porcine epidemic diarrhea virus (Alphacoronavirus) to identify conserved host restriction factors. We identified glycosylphosphatidylinositol (GPI) biosynthesis as the pan-coronavirus host factor that restrict viral entry by disrupting spike protein-mediated membrane fusion at both endosomal and plasma membranes. GPI biosynthesis generates GPI moieties that covalently anchor proteins (GPI-anchored proteins [GPI-APs]) to the cell membrane, playing essential roles in various cellular processes. Through focused CRISPR knockout screens targeting 193 GPI-APs, we identified LY6E as the key downstream effector mediating the antiviral activity of the GPI biosynthesis pathway. These findings reveal a novel role for GPI biosynthesis as a conserved host defense mechanism against coronaviruses and highlight LY6E as a critical antiviral effector. This study provides new insights into virus-host interactions and the development of host-directed antiviral therapies.

microbiology↗

bioRxiv2025

A Generalist Intracortical Motor Decoder

Mapping the relationship between neural activity and motor behavior is a central aim of sensori-motor neuroscience and neurotechnology. While most progress to this end has relied on restricting complexity, the advent of foundation models instead proposes integrating a breadth of data as an alternate avenue for broadly advancing downstream modeling. We quantify this premise for motor decoding from intracortical microelectrode data, pretraining an autoregressive Transformer on 2000 hours of neural population spiking activity paired with diverse motor covariates from over 30 monkeys and humans. The resulting model is broadly useful, benefiting decoding on 8 downstream decoding tasks and generalizing to a variety of neural distribution shifts. However, we also highlight that scaling autoregressive Transformers seems unlikely to resolve limitations stemming from sensor variability and output stereotypy in neural datasets. Code: https://github.com/joel99/ndt3

neuroscience↗

bioRxiv2025

The single amino acid change of R516K enables efficient generation of vesicular stomatitis virus-based Crimean-Congo hemorrhagic fever reporter virus

Crimean-Congo hemorrhagic fever virus (CCHFV) is a medically important tick-borne virus, causing severe hemorrhagic diseases in humans. There is no approved vaccines and therapeutics for CCHFV infection. The study of CCHFV authentic virus requires biosafety level 3 facilities, hindering the research and development of antivirals. Here we report the generation of a recombinant vesicular stomatitis virus (VSV) bearing both CCHFV glycoprotein precursor (GPC) and EGFP reporter (rVSV-CCHFV-GFP). We also find that the acquisition of an unexpected single R516K mutation in the GPC protein enables the packaging of high-titer pseudotyped particles. The replication-competent rVSV-CCHFV-GFP reporter virus resembles the entry properties of authentic virus and allows for rapid assessment of susceptible cell lines, neutralizing antibodies, and host entry factors such as heparan sulfate in fluorescence-based assays. This study provides valuable strategy for packaging of high-titer CCHFV pseudovirus, and the tool generated here can be served for the identification and evaluation of countermeasures against the cell entry of CCHFV.

microbiology↗

bioRxiv2025

Melatonin attenuates kidney injury by alleviating lysosomal damage in diabetic kidney disease

Proteinuria-induced damage to renal tubular epithelial cells is one of the main causes of diabetic kidney disease (DKD), and the clearance of overloaded albumin by lysosomes is crucial for maintaining the homeostasis of renal tubular epithelial cells. Therefore, lysosomal damage is closely related to the pathogenesis of DKD, but effective prevention and treatment measures are still lacking. Melatonin (MLT) is secreted by the pineal gland and can not only regulate circadian rhythms but also maintain lysosomal homeostasis. In this study, we demonstrate the presence of significant lysosomal damage in renal tubules of DKD patients, which causes autophagy impairment and a concomitant oxidative stress imbalance; however, MLT can upregulate transcription factor EB (TFEB) to improve lysosomal damage and restore the biosynthesis in this organelle. Mechanistically, MLT may protect lysosomes via the upregulation of TFEB and the miR-205-5p-LRP-1 pathway in renal tubules, thus improving autophagy dysfunction and oxidative imbalance in DKD.

pathology↗

bioRxiv2025

Absolute Quantification of Aging-Associated Glycans in IgG for Biological Age Prediction: Insights from Glycomics and Transcriptomics

Immunoglobulin G (IgG) N-glycans have been identified as associated with aging; however, previous studies predominantly quantified changes based on the relative percentages of each glycan within the total glycan pool, neglecting the absolute concentration changes of individual glycans. Additionally, relative quantification can limit practical applications, as these often require absolute concentration measurements for consistent and interpretable biomarker values. In this study, we introduce a novel strategy for discovering aging-associated IgG glycans and establishing a prediction model based on their absolute concentration alteration. We employed a glycome quantification technology to identify the alteration in IgG glycan amount in natural aging and anti-aging (caloric restriction) models, discovering aging-related glycans. The glycomics analysis revealed key features: downregulation of bisected glycan GP3 (F(6)A2B) and upregulation of digalactosylated glycan GP8 (F(6)A2G2). These glycan changes showed significant fold changes from an early stage. Using external standards of these two glycans, we subsequently measured the absolute concentrations of them, allowing us to establish a predictive model, abGlycoAge, for biological aging. The abGlycoAge index suggested a younger state under caloric restriction, with an average age reduction of 3.9-14 weeks. Additionally, we performed RNA sequencing on splenic B cells of mice, suggesting Derl3, Smarcb1, Ankrd55, Tbkbp1 and Slc38a10 could contribute to the alteration of GP3 and GP8 in aging. This analysis enhances our understanding of glycan alterations, accounts for individual variability, and aids in designing effective anti-aging strategies. These findings highlight the crucial roles of the GP3 and GP8 as potential biomarkers for aging and health.

biochemistry↗

bioRxiv2024

Elastase mediated white matter damage in cerebral small vessel disease: Microglia - neutrophils pas de deux

Cerebral small vessel disease (CSVD) leads to an extensive white matter damage associated with cognitive decline, yet the underlying damaging mechanisms remain incompletely understood. Here we established a positive correlation between plasma levels of serine proteinase elastase ELANE and periventricular white matter hyperintensity (PV-WMH) in a cohort of CSVD patients. In a CSVD murine model induced by bilateral carotid artery stenosis (BCAS), upregulated ELANE was detected both in microglia and peripheral blood neutrophils. Genetic ELANE deficiency significantly alleviated oligodendrocyte loss, thereby reducing white matter lesions (WMLs) as well as ameliorating sensorimotor and cognitive impairments in BCAS mice. In vitro studies demonstrated that ELANE triggered time-dependent and dose-dependent oligodendrocyte lineage cell death. Bone marrow transplantation showed that ELANE from microglia and peripheral blood both contributed to WML development and BCAS-induced neurological deficits. Mechanistically, ELANE, accumulated by oligodendrocytes, cleaved the phosphodiesterase domain of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Pharmacological inhibition of ELANE with Sivelestat reduced oligodendrocyte loss and WMLs leading to the restoration of white matter integrity and neurological improvements in BCAS mice. In post-mortem brain specimens of CSVD patients ELANE accumulated within WMLs being predominantly localized in microglia (and hence defined as microglial ELANE) rather than in the brain-infiltrating neutrophils. We therefore posit microglial ELANE as an instigator of whiter matter injury in CSVD and suggest its potential therapeutic relevance.

neuroscience↗

bioRxiv2024

Protocol for assessing DNA damage levels in vivo in rodent testicular germ cells in the Alkaline Comet Assay

Few protocols are available for retrieving male germ cell-specific information on DNA damage dynamics or assessing the potential harmful effects of environmental contaminants, drugs, or lifestyle factors related to genotoxicity. Here, we present a protocol for evaluating testicular germ cell genotoxicity using a modified version of the alkaline comet assay in rodent testicular germ cells. The protocol includes experimental design, preparation of testicular cell suspensions, comet analysis, germ cell-specific scoring, and data curation methods to collect information on testicular male germ cells, specifically spermatids and primary spermatocytes. For complete details on the use of this protocol please refer to Olsen et al., in preparation HighlightsO_LIDNA damage levels can be specifically measured in testicular germ cells using the developed revised version of the alkaline comet assay C_LIO_LI1C spermatids as well as 4C primary spermatocytes can be assessed C_LIO_LIBoth manual- and modeling-based approaches were developed that facilitate user-friendly protocols to select 1C spermatid comets. C_LIO_LIThis protocol expands the limited methodologies available to study germ cell DNA damage dynamics C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/624648v7_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c1e54eorg.highwire.dtl.DTLVardef@b569e9org.highwire.dtl.DTLVardef@3c74adorg.highwire.dtl.DTLVardef@110122e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

pharmacology and toxicology↗

bioRxiv2024

Identification of Differentially Expressed Genes and Proteins Related to Diapause in Lymantria Dispar: Insights for the Mechanism of Diapause from Transcriptome and Proteome Analyses

Spongy moth (Lymantria dispar Linnaeus) is a globally recognized quarantine leaf-eating pest. Spongy moths typically enter diapause after completing embryonic development and overwinter in the egg stage. They spend three-quarters of their life cycle (approximately nine months) in the egg stage, which requires a period of low-temperature stimulation to break diapause and continue growth and development. In this study, we explored the molecular mechanism underlying the diapause process in spongy moth. We performed bioinformatics analysis on four Asian populations of spongy moth and one Asian-European hybrid population through a transcriptome analysis combined with proteomics. The results revealed that 1,842 genes were differentially expressed upon diapause initiation, while 264 genes were identified upon diapause termination. Eight diapause-related genes were screened out from the three-level pathways that were significantly enriched by differentially expressed genes at the time of diapause and diapause termination, and the phylogenetic tree and protein three-dimensional structure model were constructed. This study elucidates the diapause mechanism of spongy moth at the gene and protein levels, providing theoretical insights into the early and precise prevention and control of spongy moth. This study can facilitate the development of an efficient, environmentally friendly control system for managing spongy moth populations in the field.

genomics↗

bioRxiv2024

CEACAM1 Targets ABCA1 to Mediate Atrial Fibrillation via Lipophagy-Dependent Ferroptosis

Atrial fibrillation (AF) is one of the most common arrhythmias, imposing a significant burden on individuals due to its association with cerebrovascular events. The mechanisms underlying AF involve cardiac structural remodeling, electrical remodeling, and metabolic remodeling, with oxidative stress playing a crucial role. Our previous studies demonstrated that upregulation of CEACAM1 can increase oxidative stress, inhibit cell proliferation, and potentially contribute to cellular damage, while ferroptosis is closely associated with AF. In this study, we observed a significant increase in CEACAM1 expression in an AF model. Knocking out CEACAM1 revealed changes in mitochondrial membrane potential, reactive oxygen species (ROS) levels, intracellular Ca2+ concentration, channel proteins, and glycolipid metabolic enzymes. This knockout reversed electrical, structural, and metabolic remodeling in AF. Further investigation showed that CEACAM1 knockout inhibited ferroptosis, as evidenced by ferroptosis-related indicators. Using colocalization analyses of lipid droplets, lysosomes, and autophagosomes, we discovered that CEACAM1 knockout suppressed autophagy and lipophagy during AF. Supplementation with free fatty acids (FFAs) in the AF model post-CEACAM1 knockout suggested that the inhibition of ferroptosis was linked to a reduction in FFAs. Transcriptomic and non-targeted metabolomic analyses, along with ABCA1 interference studies, indicated that CEACAM1 knockout mitigates AF by promoting ABCA1 expression. In summary, CEACAM1 targets ABCA1 to alleviate AF through the regulation of lipophagy-dependent ferroptosis, as demonstrated in both cellular and animal models. BackgroundFerroptosis is a regulated form of cell death, but its connection with atrial fibrillation(AF) remains unclear. Since CEACAM1 knockout reduces oxidative stress and protects cells, we investigated the relationship among CEACAM1, ferroptosis, and AF to uncover the regulatory mechanisms of ferroptosis in AF. MethodsWe established AF models using HL-1 cells, human pluripotent stem cells, and rats. CEACAM1 expression levels were assessed in these models, along with changes in mitochondrial membrane potential, ROS levels, intracellular Ca2+ concentration, channel proteins, glycolipid metabolic enzymes, and ferroptosis-related indicators following CEACAM1 knockout. Through transcriptomics, non-target metabolomics, and immunoprecipitation screening, we identified ABCA1 as a downstream gene of CEACAM1 involved in protecting against AF. Subsequently, ABCA1 interference experiments were conducted, and colocalization analyses of lipid droplets, lysosomes, and autophagosomes were performed after the application of ferroptosis activators, lipophagy inhibitors, and lipophagy activators. FFAs were added to the AF model to evaluate changes in lipophagy and ferroptosis following CEACAM1 knockout. ResultsIn the AF model using HL-1 myocytes, CEACAM1 knockout reverse electrical, structural, and metabolic remodeling while inhibiting ferroptosis, with ABCA1 as a downstream gene. In the AF model derived from pluripotent stem cells differentiated into atrial myocytes, CEACAM1 knockout suppressed autophagy and lipophagy during AF, thereby inhibiting ferroptosis, which correlated with reduced FFA release. This process was regulated by ABCA1. In the rat AF model, ABCA1, identified as a downstream gene of CEACAM1, was further confirmed to inhibit ferroptosis by regulating FFA levels. ConclusionThis study reveals that CEACAM1 knockout inhibits ferroptosis in AF models by upregulating ABCA1 and reducing FFA levels, providing a novel regulatory mechanism for ferroptosis in AF. O_FIG O_LINKSMALLFIG WIDTH=172 HEIGHT=200 SRC="FIGDIR/small/625561v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@809c82org.highwire.dtl.DTLVardef@1a339d6org.highwire.dtl.DTLVardef@12a20d4org.highwire.dtl.DTLVardef@d14927_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

BackgroundProtein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes, and mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L) induce protein aggregate pathology with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. We hypothesized that mitochondrial uptake of cytosolic protein aggregates and their removal by mitophagy, a lysosomal degradative pathway essential for myocardial homeostasis, facilitates cytosolic protein quality control in cardiac myocytes. MethodsMice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assessed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Induced pluripotent stem cell (iPSC)-derived cardiac myocytes with R120G knock-in to the CRYAB locus were assessed for localization of the CRYAB protein. Transgenic mice expressing R120G CRYAB protein (R120G-TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one Traf2 allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. ResultsCardiomyocyte-specific ablation of TRAF2 results accumulation of mitochondrial and cytosolic protein aggregates and DESMIN mis-localization to protein aggregates. Isolated mitochondria take up cardiomyopathy-associated aggregate-prone cytosolic chaperone proteins, namely arginine to glycine (R120G) CRYAB mutant and proline to lysine (P209L) BAG3 mutant. R120G-CRYAB mutant protein increasingly localizes to mitochondria in human and mouse cardiomyocytes. R120G-TG mice demonstrate upregulation of TRAF2 in the mitochondrial fraction with increased mitophagy as compared with wild type. Adult-onset inducible haplo-insufficiency of TRAF2 resulted in accelerated mortality, impaired left ventricular systolic function and increased protein aggregates in R120G-TG mice as compared with controls. Conversely, AAV9-mediated TRAF2 transduction in R120G-TG mice reduced mortality and attenuated left ventricular systolic dysfunction, with reduced protein aggregates and restoration of normal localization of DESMIN, a cytosolic scaffolding protein chaperoned by CRYAB, as compared with control AAV9-GFP group. ConclusionsTRAF2-mediated mitophagy in cardiac myocytes facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.

cell biology↗

bioRxiv2024

Divergent effects of 3-nitrooxypropanol on enteric methane emissions in Holstein and Brown Swiss cows, and its lack of synergy with Acacia mearnsii tannin extract

The objectives of this study were to investigate the combined effects of 3-nitrooxypropanol (3-NOP) and Acacia mearnsii tannin extract (TAN), and their interactions with dairy cattle breed [Brown Swiss (BS) vs. Holstein Friesian (HF)], on lactational performance, and enteric methane (CH4) emissions. Sixteen multiparous mid-lactation cows, including 8 BS and 8 HF cows were used in a split-plot design, with breed as the main plot. Cows within each subplot were arranged in a replicated 4 x 4 Latin Square design with a 2 x 2 factorial arrangement of treatments across four 24-d periods. The experimental diets were: 1) CON (basal total mixed ration), 2) 3-NOP (60 mg/kg DM), 3) TAN (3% of DM), and 4) 3-NOP + TAN. Spot samples of urine, feces, and gas emissions (via GreenFeed) were collected at the end of each period 8 times over 3 days. No 3-NOP x TAN x Breed interactions were observed for DM intake (DMI), milk production, or enteric gas emissions, except for CH4 yield (g/kg DMI) and CO2 production. Breed influenced DMI, milk production, and component yields, with HF cows consuming 3.7 kg/d more DMI, producing 9.3 kg/d more milk, and achieving greater feed efficiency and higher milk component yields than BS cows. Milk yield and energy-corrected milk (ECM) tended to increase in HF but tended to decrease in BS cows by 3-NOP. Cows fed TAN had 1 kg/d lower DMI with the tendency for 3-NOP x TAN showed greater reduction when TAN was fed alone, but milk yield, ECM, and feed efficiency remained unchanged. Cows fed TAN exhibited 18% lower milk urea nitrogen (N) concentration and 23.0% lower urinary N but 36.7% greater fecal N excretions as a percentage of daily N intake. A 3-NOP x Breed interaction was observed in CH4 production (g/d), with a 21.7% reduction in HF, and a 13.0% reduction in BS. Similarly, there were 3-NOP x Breed tendencies in CH4 yield and intensity (g/kg ECM), with reductions in HF cows of 21.8% and 23.4%, respectively, compared to 11.0% and 10.8% in BS cows. In conclusion, there were no synergistic or additive effects between 3-NOP and TAN on enteric CH4 mitigation. The enteric CH4 emission mitigating effect of 3-NOP was more pronounced in HF cows than in BS cows. Further research is needed to understand breed-specific responses and to optimize CH4 mitigation strategies for inclusion in national greenhouse gas inventories. ImplicationsThis study demonstrated that the feed additive 3-nitrooxypropanol reduces enteric methane emissions in dairy cows, with a greater reduction observed in Holsteins (22%) compared to Brown Swiss (13%) cows. While combining 3-NOP with Acacia mearnsii tannin extract did not further reduce methane. Feeding Acacia mearnsii extract decreased nitrogen excretion, potentially reducing environmental nitrogen pollution from manure. Neither additive substantially impacted milk production; however, 3-nitrooxypropanol tended to increase milk yield in Holsteins while reducing it in Brown Swiss. Overall, this study suggests that enteric methane mitigation efficacy of dietary strategies for different cattle breeds should be further investigated.

animal behavior and cognition↗

bioRxiv2024

Severe traumatic brain injury temporally affects cerebral blood flow, endothelial cell phenotype, and cilia

BackgroundPrevious clinical work suggested that altered cerebral blood flow (CBF) in severe traumatic brain injury (sTBI) correlates with poor executive function and clinical outcome. However, the molecular consequences of altered CBF on endothelial cells (ECs) and their blood flow-sensor organelle called cilia are not known. MethodsWe performed laser speckle contrast imaging, single cell isolation, and single cell RNA sequencing (scRNAseq) after sTBI in a closed skull, linear impact mouse model. Validation of select ciliary target protein changes was performed using flow cytometry. Additionally, in vitro experiments modeled the post-injury hypoxic environment to evaluate the effect on cilia protein ARL13B in human brain microvascular ECs. ResultsWe detected immediate reductions in CBF that were sustained for at least 100 minutes in both impacted and non-impacted sides of the brain. Our scRNAseq data detected heterogeneity in the brain cortex-derived EC cluster and demonstrated that two of five unique EC sub-clusters changed their relative proportions post-sTBI. Consistent with flow changes, we identified multiple genes associated with the fluid shear stress pathway that were significantly differentially expressed in brain ECs post-injury. Also, ECs displayed activation of ischemic pathway as early as day 1 post-injury, and enrichment of hypoxia pathway at day 7 and 28 post- injury. Arl13b ciliary gene expression was lost on day 1 in ECs cluster and remained lost for the entire course of the injury. We validated the loss of cilia protein ARL13B specifically from brain ECs as early as day 1 post-injury and detected the protein in the peripheral blood of the injured mice. We also determined that hypoxia could induce loss of ARL13B protein from cultured ECs. ConclusionsIn severe TBI, blood flow is disrupted in both impacted and non-impacted regions of the brain, creating a hypoxic environment that may influence ciliary gene and protein expression on ECs.

neuroscience↗