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Results for “pharmacology and toxicology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2026

Novel Dissymmetric Ionizable Lipid-Assembled Lipid Nanoparticles for Delivery of Ferroptosis-Related siRNA in Diabetic Treatment

Small interfering RNA (siRNA) enables precise post-transcriptional gene silencing for refractory diseases, yet its clinical translation remains limited by the lack of safe and efficient delivery vectors. Inspired by the dissymmetric alkyl chain architecture of natural membrane phospholipids, we designed and synthesized 34 novel ionizable lipids with dissymmetric hydrophobic tails and formulated them into lipid nanoparticles (LNPs). Through systematic physicochemical and biological assessments, we established clear structure-activity relationships and identified two lead LNPs (O14-LNP, H18a-LNP) with superior endosomal escape capacity, enhanced in vivo gene silencing potency, and favorable biosafety relative to the clinical benchmark MC3-LNP. In both streptozotocin-induced and spontaneous db/db type 2 diabetes (T2D) mouse models, lead LNPs delivering ferroptosis-related siRNAs effectively ameliorated glucose and lipid metabolic disorders, restored islet function, and alleviated hepatic steatosis. This study not only lays a theoretical foundation for the rational design of novel ionizable lipids, but also validates the therapeutic potential of siRNA therapy targeting ferroptosis, providing a versatile delivery platform and targeted therapeutic strategy for the treatment of T2D.

pharmacology and toxicology↗

bioRxiv2026

Lipid-ASO therapeutics exhibit differential tissue targeted delivery upon systemic or local CNS administration

Antisense oligonucleotides (ASOs) are a powerful therapeutic modality, but their full potential is hindered by pharmacokinetic properties that affect tissue and cellular delivery. Lipid conjugation is increasingly used to modulate ASO's biodistribution and promote extrahepatic activity, yet lipid dependent effects on in vivo functional delivery, particularly in the central nervous system (CNS), remain less explored. Here, we performed a side by side in vivo comparison of cholesterol, palmitic acid (C16:0), docosanoic acid (C22:0), and eicosapentaenoic acid (C20:5) conjugated to a fully phosphorothioated 3 10 3 LNA gapmer ASO targeting the Malat1 long non coding RNA. Lipid-ASO conjugates were administered systemically or locally in the brain of mice and evaluated for tissue level and cellular level distribution by imaging, qPCR and single-cell RNA sequencing, simultaneously annotating cell origin and global transcriptional changes within the cell. Following systemic administration in mice, lipid conjugation improved overall multi organ efficacy compared to unconjugated ASO, but with pronounced tissue specific differences. Single cell sequencing of liver and heart transcriptomes revealed lipid dependent cellular uptake patterns and transcriptional responses distinct from administration of unconjugated ASO. After intracerebroventricular administration, selected fatty acid conjugates enhanced silencing in deep brain regions such as the striatum, whereas cholesterol conjugation impaired functional delivery despite increased CNS retention. Light-sheet microscopy showed restricted parenchymal penetration of cholesterol ASOs compared with broader but heterogeneous distribution of palmitic acid conjugate. Together, these findings demonstrate that lipid identity critically determines ASO efficacy, productive cellular uptake, and regional CNS engagement, emphasizing the need for context specific lipid design in ASO therapeutic development.

pharmacology and toxicology↗

bioRxiv2026

Characterization and pharmacological modulation of Alzheimers disease-associated human microglial states

Microglia are central mediators of Alzheimers disease (AD) pathogenesis, yet the mechanisms driving disease-associated microglial states and their therapeutic modulation remain poorly understood. Here, we integrated single-nucleus transcriptomic datasets across the AD spectrum and identified disease- and lipid-associated microglia (DLaM) as a major AD-enriched population linked to genetic risk, neuropathology and cognitive decline. To model this state experimentally, we screened AD-relevant perturbations in human induced pluripotent stem cell (hiPSC)-derived microglia and found that ferric ammonium citrate (FAC) reproducibly induced a DLaM-like state characterized by lipid accumulation, lysosomal dysfunction and impaired A{beta} phagocytosis. Using a transcriptomics-based state-reversion screen, we identified LY2090314 as a potent modulator that restored microglial function and induced a distinct lysosomal-metabolic state. These findings establish a framework for transcriptomic disease-state-guided therapeutic discovery in AD.

neuroscience↗

bioRxiv2026

Development and pharmacological evaluation of an intranasal liposomal norbinaltorphimine formulation for the prevention of pain-induced negative affect

Chronic pain is frequently accompanied by negative affect and motivational deficits due to dysregulated mesocorticolimbic dopamine and kappa opioid receptor (KOR) signalling. Although intracranial KOR antagonism prevents pain-induced negative affect in preclinical models, systemic KOR antagonists can produce adverse off-target effects in the periphery, thereby limiting its clinical utility. Consistent with this, we found that systemic administration of KOR antagonist norbinaltorphimine (NorBNI), exacerbated motivational deficits in rats with persistent inflammatory pain. We hypothesized that maximizing central and minimizing peripheral KOR antagonism could overcome these limitations. To test this, we engineered an intranasal liposomal NorBNI formulation incorporated into an in-situ forming mucoadhesive hydrogel to enable selective nose-to-brain delivery (Nor-BNILV-HG). We characterized its physicochemical properties and functional efficacy in rats with inflammatory pain produced by Complete Freund's Adjuvant (CFA). NorBNI-loaded liposomes exhibited high drug entrapment efficiency, nanometric size, and suitable surface charge for intranasal administration. The selected thermosensitive hydrogel demonstrated appropriate gelation properties and sustained drug release. Intranasal administration of NorBNI-LV-HG produced negligible systemic NorBNI levels compared with intraperitoneal delivery. In vivo microdialysis showed that NorBNI-LV-HG prevented KOR agonist-induced reductions in nucleus accumbens (NAc) dopamine release, confirming functional central KOR blockade. Behaviourally, intranasal NorBNI-LV-HG attenuated pain-induced impairments in sucrose motivation. Importantly, unlike systemic NorBNI, repeated intranasal NorBNI-LV-HG did not alter mechanical nociceptive thresholds in pain-naive animals, suggesting this strategy mitigates unwanted peripheral nociceptive effects. Together, these findings demonstrate that intranasal NorBNI-LV-HG achieves functional brain KOR antagonism while minimizing systemic exposure and off-target effects. Selective nose-to-brain delivery of KOR antagonists therefore represents a promising therapeutic strategy to prevent and potentially reverse the affective and motivational consequences of pain and may overcome key translational barriers associated with systemic KOR treatments.

neuroscience↗

bioRxiv2026

Heterogeneous and conserved radiation responses reveal FOXM1-dependent regulation of microcephaly genes in glioblastoma

Glioblastoma (GBM) is characterized by marked heterogeneity, glioma stem-like cells (GSCs), and resistance to therapy. Because GSCs share features with neural progenitor cells (NPCs), we investigated whether neurodevelopmental programs contribute to their response to irradiation. Transcriptional profiling of four patient-derived GSC lines revealed cell line-specific responses, with radiosensitivity correlating with the magnitude of p53 activation and basal expression of its negative regulator, MDM2. Despite this heterogeneity, radiation consistently activated p53-dependent pathways and suppressed cell-cycle programs. Among these, genes associated with primary hereditary microcephaly (MCPH) that regulate NPC proliferation were coordinately repressed. Single-cell RNA sequencing localized this response to G2/M-cycling cells. FOXM1 was similarly reduced following irradiation, emerged as a candidate regulator of a subset of MCPH genes, and correlated with their expression in GBM tumors. Pharmacological inhibition of FOXM1 reduced expression of selected MCPH genes and enhanced radiosensitivity in U251 cells. Together, these findings identify coordinated suppression of a FOXM1-associated MCPH program as part of the GBM radiation response, while suggesting that the radiosensitizing effects of pharmacological FOXM1 inhibition extend beyond this transcriptional axis.

cancer biology↗

bioRxiv2026

Hypothermic Conditions Impair GnRH Pulse Generator Activity and Gametogenesis

Mammalian reproductive function is driven by arcuate kisspeptin neurons, pacemakers of gonadotropin secretion. During energy shortages, animals reallocate resources from reproduction to survival; however, the underlying neural mechanisms remain elusive. Here we used fiber photometry to chronically monitor synchronized episodes of arcuate kisspeptin neuron activity (SEskiss) in adult mice under various energy-saving conditions. In both sexes, SEskiss frequency was markedly suppressed during fasting-induced torpor and pharmacologically induced hypothermia, whereas hypometabolism alone had no discernible effect. A Q neuron-induced hypothermic state (QIH) robustly suppressed SEskiss, leading to impaired gamete maturation, whereas warming the body temperature during QIH fully restored SEskiss frequency. These findings demonstrate that hypothermia, rather than hypometabolism, is the primary driver of suppression of the hypothalamic reproductive axis during energy-saving conditions. This study provides insights into how thermal signals act as critical gatekeepers in the mammalian reproductive system.

neuroscience↗

bioRxiv2026

Ex vivo glioblastoma migration phenotypes define clinical recurrence and tumor heterogeneity

Glioblastoma's pronounced migratory capacity underlies its diffuse invasion, presenting a formidable barrier to successful treatment. Ex vivo characterization of glioblastoma cells isolated from freshly resected clinical samples under physiologically relevant conditions revealed two distinct migratory phenotypes, Fast Migrating (FM) and Slow Migrating (SM). These phenotypes reflect distinct mechanosensitivity profiles and are associated with pharmacological responses that support the motor clutch model of cell migration. Analysis of genes associated with these phenotypes revealed a transcriptomic signature that closely associated with in vitro cell migration, histological invasion in patient specimens, and clinical survival. Single-nucleus RNA sequencing revealed that FM and SM cells coexist within a single glioblastoma, with FM cells enriched at the periphery and SM cells localized to the tumor core. Collectively, our study demonstrates the utility of ex vivo glioblastoma characterization, allowing decoding of tumor heterogeneity and clinical prognostication as well as providing a framework for deconvoluting the complex cancer phenotype.

cancer biology↗

bioRxiv2026

Nuclear Myosin VI stabilises Ku-associated DNA ends during non-homologous end joining

DNA double-strand breaks (DSBs) require rapid signalling and physical stabilisation of broken DNA ends to preserve genome integrity. Here, we identify myosin VI (MVI) as an ATM-regulated component of the DSB response. DNA damage induces rapid nuclear accumulation and nanoscale reorganisation of MVI across multiple cell models, in an ATM-dependent manner. Pharmacological or genetic perturbation of MVI attenuates {gamma}H2AX signalling and disrupts Ku80 organisation, while DNA damage persists. This leads to increased sensitivity to cisplatin and bleomycin. Super-resolution imaging reveals spatial association of MVI with Ku80-containing repair structures, implicating MVI in non-homologous end joining (NHEJ). In a minimal reconstituted system, MVI and actin enhance the proximity of Ku70/80-bound DNA ends. Together, our findings identify MVI as a regulator of DSB repair that links ATM signalling to Ku-associated DNA-end stabilisation and suggest that targeting MVI may sensitise tumour cells to genotoxic therapy.

cancer biology↗

bioRxiv2026

GDF15 contributes to inflammasome-associated excessive mechanoresponses of hyperlipidemic PdL fibroblasts

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.

cell biology↗

bioRxiv2026

MAPT regulates autophagic-lysosomal function and phagocytosis in human microglia

Tauopathies are characterized by the accumulation and spread of pathogenic tau aggregates throughout the brain, a process that is increasingly recognized to involve not only neurons but also microglia. However, whether pathogenic MAPT directly alters microglial degradative capacity remains poorly understood. Here, using isogenic human induced pluripotent stem cell-derived microglia carrying the pathogenic MAPT IVS10+16 mutation, we identify tau as a regulator of microglial lysosomal function. MAPT IVS10+16 microglia exhibited coordinated suppression of lysosomal and autophagic pathways, reduced lysosomal protease abundance and activity, and impaired autophagosome-lysosome fusion. Mutant microglia also showed reduced uptake of extracellular tau aggregates, reduced tau accumulation in acidic compartments, and a blunted lysosomal response to proteopathic stress. Conversely, genetic loss of MAPT increased lysosomal degradative capacity and accumulation of extracellular tau aggregates within acidic compartments, supporting a cell-intrinsic role for endogenous tau in regulating microglial degradative function. Pharmacologic enhancement of the autophagy lysosome pathway in MAPT IVS10+16 microglia increased proteolytic activity and improved tau handling. Together, these findings reveal a reciprocal relationship between tau and microglial lysosome function and identify degradative capacity as a modifiable component of the microglial response to tau pathology.

neuroscience↗

bioRxiv2026

GABAB Receptors Gate Sex-Specific Synaptic Plasticity in the Nucleus Accumbens

Excitatory synaptic plasticity within the nucleus accumbens (NAc) drives motivated behaviors, and dysregulation is implicated in several psychiatric disorders marked by impaired reward processing. The NAc integrates glutamatergic input, which conveys information about reward, context, and behavioral goals, with local GABAergic signaling that regulates excitatory transmission and medium spiny neuron (MSNs) output. However, little is known regarding GABA-dependent modulation of activity-dependent excitatory synaptic plasticity. Here, we investigated GABAB receptor (GABABR) regulation of plasticity at hippocampus (Hipp)-NAc synapses, at which plasticity is a key mediator of reward-related behaviors. Using whole-cell electrophysiological recordings in mouse brain slices, we found that pharmacological inhibition of GABABRs converts long-term potentiation (LTP) into long-term depression (LTD) selectively in females, identifying a sex-specific role for GABABRs in modulating long-term plasticity of Hipp-MSN synapses. This LTD required mGluR5 activation and estrogen receptor alpha (ER) in both D1- and D2-expressing MSN subtypes, while only D1-MSNs suggested that LTD was expressed presynaptically through a CB1 receptor-dependent mechanism. Notably, GABABR inhibition did not alter basal synaptic transmission, indicating a specific role for these receptors in gating plasticity beyond regulation of basal excitatory drive. Together, these findings identify a novel, sex-specific mechanism by which GABABRs control the direction of synaptic plasticity.

neuroscience↗

bioRxiv2026

Cryo-EM Structure of a Triazole alpha-Conotoxin GI Mimetic Bound to the Muscle-Type Nicotinic Acetylcholine Receptor

Disulfide-rich peptides possess exceptional potency and selectivity but are often limited by the instability and synthetic challenges associated with native disulfide bonds. Here, we report the design, synthesis, pharmacological evaluation, and structural characterisation of triazole-based peptidomimetics of the -GI conotoxin, a selective antagonist of the muscle-type nicotinic acetylcholine receptor (nAChR). A series of 1,4- and 1,5-disubstituted triazole analogues were prepared entirely on resin using CuAAC and RuAAC chemistry to replace the native Cys3/13 disulfide bridge. Functional evaluation against human muscle nAChRs revealed that 1,5-triazole analogues retained low-nanomolar potency, with the lead mimetic exhibiting activity comparable to native -GI. Cryo-electron microscopy of the lead compound bound to the muscle-type nAChR provided the first structure of a disulfide-isostere peptidomimetic in complex with a membrane receptor. The structure demonstrates that the 1,5-triazole reproduces the native peptide fold with high fidelity while contributing receptor-facing interactions not available to the native disulfide bridge. Molecular dynamics simulations further revealed conserved hydration networks and similar conformational sampling between the native peptide and lead mimetic. Together, these findings establish triazoles as effective disulfide surrogates and provide a structural framework for the rational design of stabilised conotoxin therapeutics.

biochemistry↗

bioRxiv2026

Personalized phosphoproteomics establish mTORC1 as a regulator of exercise-induced insulin sensitization in human skeletal muscle

Exercise enhances skeletal muscle insulin sensitivity, but the signaling mechanisms responsible are poorly understood. Understanding them may open new therapeutic avenues for individuals with limited exercise capacity. Here, we used rapamycin to inhibit mTORC1 in combination with exercise and insulin stimulation in healthy men. A single dose of rapamycin enhanced the insulin-sensitizing effect of exercise by 53% on average compared to placebo. Responses varied widely across individuals (-40% to 218%), and we leveraged this variance through personalized phosphoproteomics to map the mTORC1-dependent signaling network in skeletal muscle. This identified the protein kinase MKNK2 as a candidate downstream effector, which we then targeted for functional validation. Pharmacological inhibition of MKNK2 with eFT508 in insulin-clamped mice reduced both whole-body and skeletal muscle insulin sensitivity, confirming a functional role for MKNK2 activity in muscle glucose uptake. We then used eFT508 in ex vivo incubated human skeletal muscle to map the signaling network downstream of MKNK2, identifying the translational initiator eIF4G1 as a further regulatory node. Together, these findings indicate that exercise-induced insulin sensitization is actively constrained by a negative feedback pathway running from mTORC1 through the translational regulators MKNK2 and eIF4G1, raising the possibility that rapid translation of unidentified target proteins contributes to fine-tuning glucose uptake.

physiology↗

bioRxiv2026

Linoleic Acid-Lyso PG Axis promoting lipid droplet-mitochondria tethering by stabilizing Noncanonically Mitochondrial PPAR β/δ to Ameliorate Microglial Dysfunction in subarachnoid hemorrhage

Background Microglial lipid handling and mitochondrial failure contribute to brain injury after subarachnoid hemorrhage (SAH), but the lipid signals coupling these processes remain unclear. We investigated whether linoleic acid (LA) restores microglial homeostasis through lysophosphatidylglycerol 16:0 (LPG[16:0]) and peroxisome proliferator-activated receptor-{delta} (PPAR{delta}). Methods Cerebrospinal fluid metabolomics included 30 patients with aneurysmal SAH and 10 control participants. Mechanisms were examined in a blood-injection mouse model and hemoglobin-exposed primary mouse microglia using targeted lipidomics, RNA sequencing, mitochondrial and phagocytosis assays, pharmacological perturbation, fractionation, coimmunoprecipitation, thermal shift analysis, and structural modeling. Behavioral outcomes were evaluated by open-field, Y-maze, and Morris water-maze testing. Results; CSF LA was higher in SAH and discriminated the groups within this cohort (area under the curve, 0.9967 [95% CI, 0.9859-1.000]; P<0.001). LA attenuated inflammatory activation and restored phagocytosis, mitochondrial membrane potential, respiration, and ATP production in hemoglobin-exposed microglia. LA restored PLA2G15-associated LPG(16:0), which phenocopied these effects. Transcriptomic and inhibitor analyses identified PPAR{delta} as a downstream effector. LPG(16:0) increased PPAR{delta} stability, and fractionation and protease protection identified a PPAR{delta} pool on the cytosolic face of the outer mitochondrial membrane. PPAR{delta} associated with PLIN2 and CPT1A, promoted lipid droplet-mitochondria apposition, and supported fatty acid oxidation. In mice, LA reduced neuroinflammatory injury and partially improved anxiety-related behavior and spatial memory.

neuroscience↗

bioRxiv2026

HIF1A recruits primate-specific endogenous retroviruses into the human hypoxic and immune responses

Oxygen availability varies profoundly across the human body and changes further during inflammation, infection, tissue injury and disease. Immune cells must therefore continuously adapt their transcriptional and metabolic state based on the oxygen availability to them. Hypoxia-inducible factor 1 (HIF1A) is central to this adaptation and a marker of the cellular response to low oxygen, yet its genomic targets have been assembled from a non-repetitive fraction of the genome, leaving nearly half of the human genome largely unexplored. Here we define the gene and transposable-element (TE) landscape of the human hypoxic response across different human tissues, cell lines, and conditions. This directional TE response was reproduced in transformed cells and in primary immune cells isolated from blood and the physiologically oxygen-restricted tonsil. Single-cell profiling of peripheral blood mononuclear cells (PBMC) under hypoxia, pharmacological HIF stabilization, and interferon stimulation revealed a striking difference between the gene and retrotranscriptome responses. While gene responses were strongly cell-type dependent and in a bidirectional manner, TEs were overwhelmingly activated. This pattern extended to blood and tonsil immune cells, where ~70-90% of tested TE families were induced under hypoxia, with activated tonsil cells showing exclusively induced significant families, including THE1B, alongside increased LTR7 and HERVH. Integrating HIF1A ChIP-seq with transcriptional responses revealed that HIF1A does not engage repetitive DNA indiscriminately. Instead, its binding converged on LTR7, the promoter long terminal repeat of the HERVH endogenous retrovirus. Approximately 80% of HIF1A-bound LTR7 elements contained a canonical hypoxia-response element, and disruption of HIF1A DNA binding dramatically reduced the expression of occupied HERVH loci. CRISPR deletion of individual LTR7/HERVH loci altered the expression of distant and neighboring genes, demonstrating that hypoxia-responsive retroelements can participate directly in host gene regulation and contribute to overall physiology. Our findings reveal the repetitive genome as a previously underappreciated component of oxygen sensing. We propose that HIF1A recruits selected endogenous retroviral elements into the human hypoxic response, extending oxygen-dependent regulation beyond conventional gene promoters and providing an additional regulatory layer through which tissue oxygenation can shape immune-cell state and human physiology.

genomics↗

bioRxiv2026

Multiscale modelling of drug-host-pathogen interaction: quantifying drug and immune contributions to treatment response

Background and Objective: Predicting treatment outcomes in infectious diseases requires accounting for the interplay between drug effects, pathogen dynamics, and host immunity. Integrating pharmacological and immunological approaches into a single simulation environment remains a fundamental challenge in both theory and practice. We aimed to develop and validate a multiscale in silico framework coupling these processes, and to quantify their respective contributions to bacterial clearance. Methods: We present the Drug-Host-Pathogen Interaction (DHPI) framework, combining three independent mechanistic components: a physiologically based pharmacokinetic model of drug disposition, a pharmacokinetic-pharmacodynamic model of drug-induced bacterial killing, and a stochastic agent-based model of the immune response. Continuous concentration profiles are time-averaged onto the agent-based time grid, assigned to bacterial phenotypic states, and converted into per-agent killing probabilities, so that drug-mediated and immune-mediated death events are recorded separately at each step. The framework was applied to simulate symptomatic pulmonary tuberculosis. Phenotype-specific drug-efficacy parameters were inferred using Approximate Bayesian Computation from historical clinical data on eight weeks of 600 mg rifampicin monotherapy, and validated against independent early bactericidal activity data over a disjoint time window. Results: The calibrated framework reproduced the observed decline in bacterial load, and matched reported early bactericidal activity over the first week. In a virtual cohort of symptomatic patients, drug-mediated killing accounted for 81-88% and immune-mediated killing for 12-19% of total bacterial elimination over the 60-day treatment course, while the dormant, granuloma-contained fraction rose from 0.20-0.29 in the first week to 0.85-0.89 at treatment completion. Over a follow-up of up to 50 years, patients reaching clinical cure had accumulated more memory lymphocytes during treatment than those progressing to clinical failure or death; moreover, the final outcome depended on the immune changes occurring during therapy rather than on the initial disease stage. Conclusions: The results show that the DHPI framework can reproduce treatment dynamics observed in patients and enable the analysis of how therapy reshapes host immune responses and subsequent disease trajectories. By explicitly representing drug-host-pathogen interactions, it provides a mechanistic basis for in silico treatment simulations and for the study of long-term immune consequences of antimicrobial therapy.

systems biology↗

bioRxiv2026

HDAC6 is a novel regulator of endothelial-to-mesenchymal transition in venous thrombosis

Background: Venous thromboembolism (VTE), which encompasses deep vein thrombosis (DVT) and pulmonary embolism (PE), is a frequent disease associated with thrombus formation and vein wall remodeling. Hence, fibrosis might result from endothelial-to-mesenchymal transition (EndMT), characterized by the loss of endothelial markers and the acquisition of mesenchymal markers. In chronic thromboembolic pulmonary hypertension, transforming growth factor (TGF{beta}), the most potent inducer of EndMT, impairs thrombus resolution. However, the molecular mechanisms implicated in TGF[beta] signaling in the context of VTE are unknown. We hypothesized that epigenetic processes regulate the TGF{beta} signaling pathway in endothelial cells promoting EndMT and vascular fibrosis. Aims: To determine if the histone deacetylase 6 (HDAC6) regulates the TGF{beta} signaling pathway in endothelial cells promoting EndMT and delays venous thrombosis. Methods: To study the role of HDAC6 in EndMT, endothelial cells were treated with a pharmacological inhibitor (TCS20b) and incubated with TGF{beta} and thrombin for 2, 3, and 5 days. Real time PCR and Western blot were performed to analyze endothelial and mesenchymal marker expression and TGF{beta} signaling. An experimental model of VTE was used to study the role of HDAC6 on thrombus size overtime. Animals were treated or not with a specific HDAC6 inhibitor (tubastatin A) for 7 to 21 days. Analysis of RNAseq data sets publicly available were used to confirm our main results. Within group and treatment differences were analyzed using two-way ANOVA and Tukeys multiple comparisons. Results: Expression of the mesenchymal markers, calponin and transgelin, was increased by TGF{beta} and thrombin. Interestingly these changes were inhibited in presence of TCS20b. TGF{beta} mediated these effects through ERK1/2 and HDAC6 activation. Inhibition of HDAC6 in vivo reduced thrombus size 7 days after surgery compared to controls. This was associated with reduced expression of the EndMT marker transgelin in endothelial cells compared to the control animals. We found that FN1-EDA expression was associated with EndMT and regulated by HDAC6 in vitro. This marker was also associated with thrombosis in the RNAseq data set that we analyzed and potentially in patients with recurrent DVT. Conclusion: We found that HDAC6 regulates EndMT in venous thrombosis and impairs thrombus resolution. HDAC6 also regulates expression FN1-EDA that appears to be a strong marker associated with DVT and DVT recurrence. Thus, HDAC6 might represent an attractive therapeutic target for patients with a high risk of recurrent VTE.

physiology↗

bioRxiv2026

Design and Validation of New Primers for Specific and Sensitive Real-time PCR Detection and Quantification of Seven Botulinum Encoding Genes (Serotype A-G) of Clostridium botulinum

Botulinum neurotoxins (BoNTs) comprise a highly diverse group of seven serotypes (from A-G) and over 40 subtypes worldwide. Previous primer- and probe-based nucleic acid amplification tests (NAATs) for detection of BoNT encoding genes are challenged by high levels of nucleotide polymorphism both across and within subtypes. In this study, multiple BoNT gene sequences were aligned to identify highly conserved regions for the design of new primers that enable the detection of all seven serotypes under the same conditions. Specific primer sets were designed and validated using in silico, conventional and real-time PCR with constructed plasmids carrying the target fragments and spiked food matrices. The established procedure achieved highly specific and sensitive detection of BoNT serotypes A-G with sensitivity of 10 copies/reaction and a total turnaround time of approximately 1.5 hours. The procedure also eliminated the carryover PCR product by using uracil-N-glycosylase in combination with dUTP in the assay reaction mix. This study provides an alternative NAAT with higher coverage and compliments the traditional mouse bioassays in enhancing global botulism surveillance capabilities.

molecular biology↗