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bioRxiv2024

Genetic makeup of Trichuris hominibus, a novel Trichuris species naturally infecting humans and displaying signs of resistance to drug treatment

BackgroundTrichuriasis is a neglected tropical disease that affects as many as 500 million individuals and can cause significant morbidity. For decades, it was thought to be caused by one species of whipworm, Trichuris trichiura. Significant differences in response rates to the best available anthelmintic treatment for trichuriasis -- a combination of albendazole and ivermectin -- exist across clinical trial reports yet the underlying reasons are unclear. MethodsCombining long- and short- read sequencing approaches, we assembled a high- quality reference genome of T. incognita n. sp. isolated in an interventional study conducted in Cote dIvoire. The species tree of the Trichuris genus was constructed using 12434 orthologous groups. We sequenced a total of 747 individual worms which were used to confirm the phylogenetic placement and investigate patterns of adaptation through comparative genomic analysis. FindingsHere, we present and characterize a human-infecting Trichuris species that we named Trichuris incognita n. sp. Comparative genomic analysis of genes suspected to confer resistance to either albendazole or ivermectin in helminths revealed a high number of beta- tubulin (TBB) orthologs, present in the whole population of T. incognita n. sp., compared to the canonical T. trichiura species, but these genes were not associated to a resistant phenotype. We conducted a genome wide association study (GWAS) comparing 179 albendazole-ivermectin sensitive to 542 drug non-sensitive worms, which did not conclusively show an adaptation to drug pressure within the same species. InterpretationOur results demonstrate that trichuriasis can be caused by multiple species of whipworm and that the differences in response rates, may be a result of species responding differently to drug treatment, as opposed to an establishment of resistance. This discovery, coupled with the high tolerability of T. incognita n. sp. to albendazole-ivermectin marks a significant shift in how we understand and approach whipworm infections. FundingEuropean Research Council (ERC) Nr. 101019223. Research in context Panel: Evidence before this studyThe recommended treatment with the benzimidazoles albendazole or mebendazole shows consistently low efficacy against T. trichiura, and therefore combination chemotherapy of benzimidazoles and ivermectin has been recommended. Recent clinical trials with albendazole-ivermectin, showed especially low cure rates against what was presumed to be T. trichiura in Cote dIvoire, in contrast to higher efficacy observed in Laos and Pemba, Tanzania. Amplicon sequencing data pointed towards underlying genomic differences and potentially a new species of human infecting Trichuris in Cote dIvoire. Added value of this studyWe identified and described a morphologically indistinguishable, novel causative agent of trichuriasis that doesnt respond to conventional drug treatment: Trichuris incognita n. sp. We provide a high-quality reference genome of this novel species and sequencing data of 747 individual isolates. A genome wide association study did not conclusively show adaptation to drug pressure. This study demonstrates that trichuriasis can be caused by multiple species of whipworm and that the differences in response rates, may be a result of species responding differently to drug treatment, as opposed to an establishment of resistance. Implications of all the available evidenceThe current whole body of evidence calls for new effective treatments against T. incognita n. sp. Due to the morphological indistinguishableness, molecular diagnostic tools need to be implemented to identify species level differences as opposed to the traditional Kato Katz method. The geographical distribution of T. incognita as well as longitudinal changes of species distributions will need to be mapped, in addition to better understanding of the new species pathology and transmission dynamics.

genomics↗
bioRxiv2023

Supporting-cell vs. hair-cell survival in the human cochlea: Implications for regenerative therapies

Animal studies have shown that the supporting-cells surviving in the organ of Corti after cochlear insult can be transdifferentiated into hair cells as a treatment for sensorineural hearing loss. Clinical trials of small-molecule therapeutics have been undertaken, but little is known about how to predict the pattern and degree of supporting-cell survival based on audiogram, hearing loss etiology or any other metric obtainable pre-mortem. To address this, we systematically assessed supporting-cell and hair cell survival, as a function of cochlear location in 274 temporal bone cases from the archives at the Massachusetts Eye and Ear and compared the histopathology with the audiograms and hearing-loss etiologies. Results showed that supporting-cell survival was always significantly greater in the apical half than the basal half of the cochlea, that inner pillars were more robust than outer pillars or Deiters cells, and that total replacement of all supporting cells with a flat epithelium was rare outside of the extreme basal 20% of the cochlea. Supporting cell survival in the basal half of the cochlea was better correlated with the slope of the audiogram than with the mean high-frequency threshold per se: i.e. survival was better with flatter audiograms than with steeply down-sloping audiograms. Cochlear regions with extensive hair cell loss and exceptional supporting cell survival were most common in cases with hearing loss due to ototoxic drugs. Such cases also tended to have less pathology in other functionally critical structures, i.e. spiral ganglion neurons and the stria vascularis. HighlightsO_LISupporting cell survival was systematically assessed in 274 human cochleas C_LIO_LISupporting cell survival was better with flat than with down-sloping audiograms C_LIO_LISupporting cell survival was most robust when hearing loss was from ototoxic drugs C_LIO_LIOtotoxic cases also showed less pathology in other critical cochlear structures C_LIO_LIThe data can inform clinical trials for regeneration via supporting cell conversion C_LI

neuroscience↗
bioRxiv2018

Characterisation of antigenic MHC Class I-restricted T cell epitopes in the glycoprotein of Ebolavirus

Ebolavirus is a pathogen capable of causing highly lethal haemorrhagic fever in humans. The envelope-displayed viral glycoprotein is the primary target of humoral immunity induced by both natural exposure and vaccination. The epitopes targeted by B cells have been thoroughly characterised by functional and structural analyses of the glycoprotein, GP, yet there is a paucity of information regarding the cellular immune response to Ebolavirus. To date, no T cell epitopes in the glycoprotein have been characterised in detail in humans.\n\nA recent Phase I clinical trial of a heterologous prime-boost vaccination regime with viral vectors encoding filovirus antigens elicited strong humoral and T cell responses in vaccinees. Using samples from this trial, the most frequently recognised peptide pools were studied in more detail to identify the minimal epitopes recognised by antigen-specific T cells and associated HLA-restrictions.\n\nUsing IFN{gamma} ELISPOT and flow cytometry, we characterised nine highly immunogenic T cell epitopes located on both the GP1 and GP2 subunits of the Ebolavirus GP. Epitope mapping revealed the location of these epitopes as presented on the mature virion. HLA-typing on all participants, combined with in silico epitope analysis, determined the likely MHC class I restriction elements. Thirteen HLA-A and -B alleles were predicted to present the identified epitopes, suggesting promiscuous recognition and induction of a broad immune response.\n\nThe glycoprotein of Ebolavirus is highly immunogenic, inducing both CD4+ and CD8+ T cell responses and we have shown here for the first time that these responses are associated with multiple HLA types. Delivery of this antigen using a heterologous prime-boost approach with ChAd3 and MVA is likely to be highly immunogenic in genetically diverse human populations, due to the induction of responses against multiple immunodominant epitopes.\n\nAuthor SummaryThe West African Ebolavirus epidemic in 2013-2016 claimed more than 11,000 lives, eclipsing all previously recorded outbreaks combined. Understanding the mechanisms of host-pathogen interactions informs rational vaccine design. B cell epitopes have been described extensively through the characterisation of monoclonal antibodies from survivors of Ebolavirus disease. Despite a clear role for cellular immunity in survival of Ebolavirus disease, data relating to minimal T cell epitopes in the glycoprotein has so far only been obtained in murine. This study describes the first identification of human T cell epitopes in the surface glycoprotein of the Ebolavirus. These epitopes have been identified using samples from a clinical trial of viral vectored vaccines against the Zaire Ebolavirus undertaken during the 2013-2016 West African outbreak. We also describe the recognition of these epitopes through multiple HLA alleles. As efforts to progress a vaccine against Ebola towards licensure proceed, detailed characterisation of immunity to the virus contribute to our understanding of the potential application of vaccines in diverse populations.

immunology↗
bioRxiv2025

Translating brain anatomy and neurodegenerative disease from mouse to human through latent gene expression space

BackgroundThe mouse model is the most widely used animal model in neuroscience, yet translating findings to humans suffers from the lack of formal models comparing the mouse and the human brain. Here, we devised a novel framework using mouse and human gene expression to build a quantitative common space and apply it to models of neurodegenerative disease. MethodsWe trained a variational autoencoder on mouse spatial transcriptomics, and embedded mouse and human gene orthologs in the models latent space. We computed a latent cross-species similarity matrix for translation and compared translated maps to human ground truth evidence. FindingsWe established the validity of our model based on anatomical homology. Independent of species, brain areas with similar latent patterns clustered together, improving the homology of known anatomical pairs, and preserving principles of brain organisation. Importantly, brain alterations in mouse disease models predicted human patterns of brain changes in Alzheimers and Parkinsons diseases. We further determined the best mouse model for the AD patients, based on how well the translations matched the patient data, across multiple models and timepoints. InterpretationOur work provides i) a quantitative bridge across evolutionary divergence between the human and the predominant preclinical species, ii) a predictive framework to help design and evaluate disease models. By highlighting which models are best suited across stages of disease, we effectively support the understanding of disease mechanisms, assist in the workflow of clinical trials, and ultimately accelerate the transformation of findings into improved human outcomes. FundingSupported by the Biotechnology and Biological Sciences Research Coundil (BBSRC) UK, the Medical Research Council (MRC) UK, the European Research Council, and the NIHR Oxford Health Biomedical Research Centre. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSInferences on the human brain based on findings in the mouse are backed by broad neuroanatomical similarities between the two species. However, interventions that were successful in rodents rarely translate into successful outcomes to treat human diseases. One way to address this challenge is to quantify the boundaries of translational possibilities using cross-species common space approaches. Low-level embedding spaces have been used to quantitatively compare across species, but there is currently no framework for effective translation of disease maps between species, and no tool to establish the best animal model for a given patient population. Added value of this studyThis study is the first to quantitatively link mouse models of neurodegenerative disease with human patient data. After validating our novel VAE-based comparative framework on anatomical homology compared to existing methods, we determine which of three Alzheimers Disease (AD) mouse models, and at which timepoint, best models a human patient population with mild AD. In addition, we predicted human brain changes in human Parkinsons Disease, based on maps from mouse models of this disease. Implications of all the available evidenceThis approach allows one to directly evaluate mouse models based on the quality of their translation to the human. At the level of preclinical model design, the translation from human to mouse of diverse disease phenotypes will highlight the relevant targets for intervention in the mouse. This will justify the relevance of specific animal models and time courses, and therefore streamline clinical trials. By additionally translating the effect of treatment or intervention longitudinally, we can optimize human treatment plans and life-long disease management. Combined with patient population stratification, this takes an important step towards personalised translational medicine.

neuroscience↗
bioRxiv2022

Lack of antiviral activity of probenecid in Vero E6 cells and Syrian golden hamsters: a need for better understanding of inter-lab differences in preclinical assays.

Antiviral interventions are urgently required to support vaccination programmes and reduce the global burden of COVID-19. Prior to initiation of large-scale clinical trials, robust preclinical data in support of candidate plausibility are required. The speed at which preclinical models have been developed during the pandemic are unprecedented but there is a vital need for standardisation and assessment of the Critical Quality Attributes. This work provides cross-validation for the recent report demonstrating potent antiviral activity of probenecid against SARS-CoV-2 in preclinical models (1). Vero E6 cells were pre-incubated with probenecid, across a 7-point concentration range, or control media for 2 hours before infection with SARS-CoV-2 (SARS-CoV-2/Human/Liverpool/REMRQ0001/2020, Pango B; MOI 0.05). Probenecid or control media was then reapplied and plates incubated for 48 hours. Cells were fixed with 4% v/v paraformaldehyde, stained with crystal violet and cytopathic activity quantified by spectrophotometry at 590 nm. Syrian golden hamsters (n=5 per group) were intranasally inoculated with virus (SARS-CoV-2 Delta variant B.1.617.2; 103 PFU/hamster) for 24 hours prior to treatment. Hamsters were treated with probenecid or vehicle for 4 doses. Hamsters were ethically euthanised before quantification of total and sub-genomic pulmonary viral RNAs. No inhibition of cytopathic activity was observed for probenecid at any concentration in Vero E6 cells. Furthermore, no reduction in either total or sub-genomic RNA was observed in terminal lung samples from hamsters on day 3 (P > 0.05). Body weight of uninfected hamsters remained stable throughout the course of the experiment whereas both probenecid- (6 - 9% over 3 days) and vehicle-treated (5 - 10% over 3 days) infected hamsters lost body weight which was comparable in magnitude (P > 0.5). The presented data do not support probenecid as a SARS-CoV-2 antiviral. These data do not support use of probenecid in COVID-19 and further analysis is required prior to initiation of clinical trials to investigate the potential utility of this drug.

pharmacology and toxicology↗
bioRxiv2026

Spindle Assembly Checkpoint Competency Determines Sensitivity to KIF18A Inhibition in Small-Cell Lung Cancer

BackgroundSmall-cell lung cancer (SCLC) is characterized by pervasive chromosomal instability (CIN) and remains largely refractory to targeted therapies. KIF18A, a motor protein that regulates chromosome alignment during mitosis, has emerged as a selective dependency in CIN-high tumors. Whether this dependency extends to SCLC, a prototypical CIN-high cancer, has not been established, and biomarkers predicting response to KIF18A inhibition, currently in clinical trials, are lacking. MethodsWe integrated analyses of patient tumor datasets, neuroendocrine (NE) and non- NE SCLC cell lines, and functional perturbation models to define the determinants of response to KIF18A inhibition. Chromosomal instability metrics, transcriptional programs, mitotic dynamics, and spindle assembly checkpoint (SAC) function were assessed using genomic profiling, live-cell imaging, genetic perturbation, and pharmacologic inhibition. ResultsKIF18A expression was elevated in SCLC tumors and correlated with CIN-associated transcriptional programs, proliferative markers, and NE status; however, these features did not predict sensitivity to KIF18A inhibition. Instead, response was determined by the functional integrity of the SAC. SAC-proficient SCLC cells underwent sustained mitotic arrest followed by apoptotic cell death upon KIF18A inhibition, whereas SAC-defective cells failed to maintain checkpoint activation and survived. Mechanistically, resistant cells exhibited impaired kinetochore recruitment of core SAC components, including MAD1 and BUBR1. Importantly, transient induction of acute CIN through MPS1 inhibition partially restored sensitivity to KIF18A inhibition in resistant models. ConclusionsThis study provides the first mechanistic characterization of KIF18A dependency in SCLC, identifying SAC competency as the primary determinant of response. These findings establish a biologically informed framework for patient stratification and rational combination strategies. Translational RelevanceSmall-cell lung cancer (SCLC) is an aggressive malignancy with few effective targeted therapies and marked chromosomal instability. KIF18A has emerged as a potential therapeutic target in genomically unstable cancers, but biomarkers predicting response to KIF18A inhibition are lacking. We demonstrate that sensitivity to KIF18A inhibition in SCLC is determined not by KIF18A expression, neuroendocrine subtype, or baseline chromosomal instability, but by the functional integrity of the spindle assembly checkpoint (SAC). SCLC cells with intact SAC signaling undergo sustained mitotic arrest and apoptosis upon KIF18A inhibition, whereas SAC-defective cells bypass checkpoint activation and survive aberrant mitosis. Notably, transient induction of acute chromosomal instability through MPS1 inhibition partially restores sensitivity in resistant models. Together, these findings identify mitotic checkpoint competency as a mechanistic determinant and candidate predictive biomarker for KIF18A-targeted therapies, providing a biologically informed framework for patient stratification and rational combination strategies relevant to ongoing KIF18A inhibitor clinical trials.

cancer biology↗
bioRxiv2025

ONC206 demonstrates potent anti-tumorigenic activity and is a potential novel therapeutic strategy for high-risk medulloblastoma

BackgroundMedulloblastoma is the most common malignant pediatric brain tumor, and has an urgent need for novel treatment approaches. Dordaviprone (ONC201) and its chemical derivative with nanomolar potency, ONC206, induce apoptosis of cancer cells by activation of the mitochondrial caseinolytic protease P (ClpP). ONC206 is currently in Phase I clinical trials for pediatric patients with primary brain tumors. MethodsIn this study, we evaluated the preclinical therapeutic effects of ONC206 in medulloblastoma and investigated its mechanism of action. ResultsWe found evidence for high expression of ClpP at both the RNA and protein level in medulloblastoma tumors, compared to very low expression in normal brain tissue. In addition, we saw a pronounced reduction in cell viability of human Group 3 and Group 4 and murine SHH-driven and Group 3 medulloblastoma cells treated with ONC206 with low IC-50s. After treatment with ONC206, we observed an induction of integrated stress response and mitochondrial damage. To test the efficacy of ONC206 in vivo, we used murine models of SHH-driven and Group 3 medulloblastoma as well as Group 3 and Group 4 patient-derived xenografts (PDXs). ONC206 led to a significant prolongation of survival in both murine models, with the SHH mice demonstrating survival extension from 70 to 140 days. PDX-bearing mice also responded to ONC206, which led to a significant survival benefit. ConclusionOur results highlight ONC206 as a novel therapeutic option for patients with high-risk medulloblastoma and provide strong rationale for testing the efficacy of ONC206 in the treatment of these patients. Key points (2-3)ONC206 potently kills medulloblastoma cells by inducing integrated stress response and mitochondrial damage. ONC206 prolongs survival of medulloblastoma-bearing mice in both murine and patient-derived xenograft models. Importance of studyThere is an unmet need for better therapies for high-risk medulloblastoma patients. ONC201 has shown promising responses and recently received FDA approval for diffuse midline glioma. ONC206 is a chemical derivative with higher potency and better brain penetrance. In this study, we analyzed the therapeutic potential of ONC206 for high-risk medulloblastoma and found that the drug effectively killed mouse and human medulloblastoma cells with high nanomolar potency. We also saw that ONC206 very significantly prolonged survival of medulloblastoma-bearing mice, both in genetically engineered mouse models and patient-derived xenografts. Our study provides a strong rationale for testing the efficacy of ONC206 in the treatment of patients with medulloblastoma and has set the stage for a clinical trial with this agent in pediatric patients with recurrent malignant brain tumors, including medulloblastoma (NCT04732065).

cancer biology↗
bioRxiv2025

Revolutionizing male contraception: Personal lubricants as a novel way to deliver the Human Contraception Antibody

Study QuestionCan personal lubricants effectively deliver the sperm-agglutinating Human Contraception Antibody (HCA) to achieve on-demand male contraception? Summary AnswerThis study demonstrates that several water-based lubricants can effectively deliver bioactive HCA, and that a dimethicone-containing silicone lubricant can be modified into a stable emulsion suitable for antibody delivery. What Is Known AlreadyThe HCA-based vaginal film ZB-06 was shown to be safe and effective in a Phase I clinical trial for female contraception. Male contraceptive options remain limited. Using sexual lubricants as a delivery platform for HCA represents a novel and practical approach to male contraception. Study Design, Size, DurationWe tested the stability of HCA for up to two years in a variety of commercially available sexual lubricants, and its delivery from a penile device using a simulated intercourse model. Participants/Materials, Setting, MethodsFourteen commercial lubricants were initially screened for impact on sperm motility and viability, miscibility with HCA solution, and preservation of HCA activity (sperm agglutination test). From these, three water-based and one silicone-based lubricant were selected for further study. The silicone-based product was engineered into a novel emulsion (KY-DE) by incorporating 6% w/w PEG-10 dimethicone and 0.005% v/v TWEEN-20 for HCA integration. Formulations were tested for HCA functional stability and contraceptive efficacy over time using kinetic sperm agglutination assays and a capillary tube sperm penetration test. A novel simulated intercourse model was developed using a 3D-printed penile device and a vaginal analog (Fleshlight(R)). HCA-lube was applied to the "penis", and concentrations of HCA delivered to the "vagina" during intercourse were measured by ELISA and sperm agglutination assay. Safety evaluations were conducted using a vaginal tissue model (EpiVaginal, VEC-100-FT). Viability (MTT assay), tight junction integrity (TEER), and cytokine release were assessed after application of HCA-lube. Main Results and the Role of ChanceAll selected lubricants were nontoxic and did not affect HCAs ability to agglutinate sperm. In the capillary tube sperm penetration assay, HCA-formulated lubricants significantly impaired sperm penetration; few motile sperm penetrated past a depth of 1 cm after 90 minutes in the HCA-lubricants vs >80_in most of the untreated lubricants. Importantly, the simulated intercourse assay confirmed successful delivery of HCA to the vagina. Water-based lubricants delivered an average of 24 {+/-} 8.4 {micro}g/mL of HCA, the commercial silicone lubricant 11 {+/-} 5.2 {micro}g/mL, and the KY-DE emulsion 27.4 {+/-} 22.4 {micro}g/mL. Futhermore, the formulations did not compromise tissue integrity or viability, or induce significant inflammatory responses. Large Scale DataNot applicable. Limitations, Reasons for CautionWhile these in vitro results support the feasibility of using lubricants as HCA delivery vehicles, translation into clinical application requires further validation and clinical trials. Wider Implications of the FindingsThis work presents a new direction in male contraceptive development, leveraging the widespread use of personal lubricants to introduce antibody-based, reversible, on-demand contraception. Engineering a silicone-based lubricant that retains HCA activity under varied conditions and ensures higher mucosal delivery could further advance this approach. Study Funding/Competing Interest(s)This work was supported by a Sokol Grant from the Male Contraceptive Initiative, a Sexual Medicine Grant from Boston University Chobanian & Avedisian School of Medicine, and grant P50HD096957 from the National Institutes of Health. Kevin Whaley (ZabBio) intends to commercialize HCA for contraception. None of the other authors have competing interests.

bioengineering↗
bioRxiv2023

A Modified BPaL Regimen for Tuberculosis Treatment replaces Linezolid with Inhaled Spectinamides

The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral- drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) (BPaL regimen) for treatment of tuberculosis (TB). This regimen achieved remarkable results as almost 90% of the multidrug resistant (MDR) or extensively drug resistant (XDR) TB participants were cured but many patients also developed severe adverse events (AEs). The AEs were associated with the long-term administration of the protein synthesis inhibitor linezolid. Spectinamide 1599 is also a protein synthesis inhibitor of Mycobacterium tuberculosis with an excellent safety profile but which lacks oral bioavailability. Here, we propose to replace L in the BPaL regimen with spectinamide (S) administered via inhalation and we demonstrate that inhaled spectinamide 1599, combined with BPa --BPaS regimen--has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the BALB/c and C3HeB/FeJ murine chronic TB efficacy models. After 4-weeks of treatment, both regimens promoted equivalent bactericidal effect in both TB murine models. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL and BPa, but no the BPaS treatment, also decreased myeloid to erythroid ratio suggesting the S in the BPaS regimen was able to recover this effect. Moreover, the BPaL also increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cells counts while increased the number of B and helper and regulatory T cells. These combined data suggest that inhaled spectinamide 1599 combined with BPa is an effective TB regimen that avoids L-associated AEs. IMPORTANCETuberculosis (TB) is an airborne infectious disease that spreads via aerosols containing Mycobacterium tuberculosis (Mtb), the causative agent of TB. TB can be cured by administration of 3-4 drugs for 6-9 months but there are limited treatment options for patients infected with multidrug (MDR) and extensively resistant (XDR) strains of Mtb. BPaL is a new all-oral combination of drugs consisting of Bedaquiline (B), Pretomanid (Pa) and Linezolid (L). This regimen was able to cure [~]90% of MDR and XDR TB patients in clinical trials but many patients developed severe adverse events (AEs) associated to the long-term administration of linezolid. We evaluated a new regimen in which Linezolid in the BPaL regimen was replaced with inhaled spectinamide 1599. In the current study, we demonstrate that 4-weeks of treatment with inhaled spectinamide 1599 in combination with Bedaquiline and Pretomanid has equivalent efficacy to the BPaL drug combination and avoids the L-associated-AEs.

microbiology↗
bioRxiv2024

Etrinabdione (VCE-004.8), a B55a activator, promotes angiogenesis and arteriogenesis in critical limb ischemia.

BackgroundVasculogenic therapies explored for the treatment of peripheral artery disease (PAD) have encountered minimal success in clinical trials. Addressing this, B55, an isoform of protein phosphatase 2A (PP2A), emerges as pivotal in vessel remodeling through activation of hypoxia-inducible factor 1 (HIF-1). This study delves into the pharmacological profile of VCE-004.8 (Etrinabdione) and evaluates its efficacy in a preclinical model of critical limb ischemia, with a focus on its potential as a PP2A/B55 activator to induce angiogenesis and arteriogenesis. MethodsVascular endothelial cells were used for in vitro experiments. Aorta ring assay was performed to explore sprouting activity. Matrigel plug-in assay was used to assess the angiogenic potential. Critical limb ischemia (CLI) in mice was induced by double ligation in the femoral arteria. Endothelial vascular and fibrotic biomarkers were studied by immunohistochemistry and qPCR. Arteriogenesis was investigated by microvascular casting and micro-CT. Proteomic analysis in vascular tissues was analyzed by LC-MS/MS. Ex-vivo expression of B55 and biomarkers were investigated in artery samples from PAD patients. ResultsVCE-004.8 exhibited the ability to induce B55 expression and activate the intersecting pathways B55/AMPK/Sirtuin 1/eNOS and B55/PHD2/HIF-1. VCE-004.8 prevented OxLDL and H2O2-induced cytotoxicity, senescence, and inflammation in endothelial cells. Oral VCE-004.8 increased aorta sprouting in vitro and angiogenesis in vivo. In CLI mice VCE-004.8 improved collateral vessel formation and induced endothelial cells proliferation, angiogenic gene expression and prevented fibrosis. The expression of B55, Caveolin 1 and Sirtuin-1 is reduced in arteries from CLI mice and PAD patient, and the expression of these markers was restored in mice treated with VCE-004.8. ConclusionsThe findings presented in this study indicate that Etrinabdione holds promise in mitigating endothelial cell damage and senescence, while concurrently fostering arteriogenesis and angiogenesis. These observations position Etrinabdione as a compelling candidate for the treatment of PAD, and potentially other cardiovascular disorders. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIThe phosphatase PPA2/B55 stabilizes endothelial cells (ECs) in response to cell stress conditions, thereby protecting ECs from apoptosis and promoting angiogenesis. C_LIO_LIEtrinabdione (VCE-004.8) functions as a potent activator of PPA2/B55 inducing PHD2 dephosphorylation at ser125 and fostering HIF activation. C_LIO_LIVCE-004.8 prevents vascular damage in preclinical models of systemic and cardiac fibrosis and alleviates blood-brain barrier disruption in neuroinflammatory conditions. C_LIO_LIVCE-004.8 is also a dual agonist of PPAR{psi} and CB2 receptors and shows antiinflammatory activity. C_LIO_LIOral VCE-004.8 has meet the primary endpoints of safety and tolerability in a Phase IIa clinical trial with systemic sclerosis patients (clinicaltrial.gov: NCT03745001). C_LI What New Information Does This Article Contribute?O_LIEtrinabdione induces HIF-1 expression in endothelial cells through a novel pathway that potentially involves two axes: B55/PHD2 and B55/AMPK/Sirt1 signaling that may converge on HIF stabilization. C_LIO_LIEtrinabdione prevented endothelial cell damage and senescence, while inducing arteriogenesis and angiogenesis in CLI mice. C_LIO_LIIn arteries of patients with PAD and in CLI mouse models, the expression levels of B55, Caveolin 1, and Sirtuin 1 are diminished. However, treatment with Etrinabdione specifically in CLI mice prompts an increase in the levels of these proteins. C_LIO_LIEtrinabdione triggers neovascularization and angiogenesis specifically within hypoxic tissue in a critical ischemia model, with no impact on healthy tissue. C_LI

pharmacology and toxicology↗
bioRxiv2026

WNT-driven chromosomal instability as a biomarker for PORCN inhibition

Targeting Porcupine (PORCN), a key regulator of the WNT-signalling pathway, has shown therapeutic potential in multiple cancers. Despite strong target engagement and acceptable safety profiles through human phase I clinical trials, low phase II efficacy has stalled further clinical development. Given that aberrant WNT signalling can drive tumorigenesis by inducing chromosomal instability (CIN), we hypothesised that genomic CIN signatures might serve as a predictive biomarker to help improve response rates. Using a controlled in vitro model and single-cell whole-genome sequencing, we demonstrate that acute WNT-activation directly induces three distinct types of CIN: whole genome duplication, replication stress, and impaired homologous recombination. We translated these observations into a composite CIN signature biomarker that significantly correlated with both genetic dependency and pharmacological inhibition of PORCN across 195 and 24 cell lines, respectively. Through a large-scale meta-analysis of patient-derived and cell line xenografts, we established that this composite CIN signature biomarker quantitatively predicts in vivo PORCN inhibitor sensitivity (R=-0.71, p<0.002). By applying an optimised biomarker threshold, refined through modelling of human patient data, to the The Cancer Genome Atlas dataset, we successfully retrospectively modelled previous trial results and identified gastroesophageal cancers as a high-prevalence (36.6%) indication for future development. We validated this strategy in a mouse clinical trial of gastric and esophageal xenografts, where biomarker-guided stratification achieved an objective response rate of 60% and significantly decreased risk of progression (HR=0.21, p=0.0345). These data establish an actionable, trail-ready framework for further PORCN inhibitor clinical development.

cancer biology↗
bioRxiv2020

Longitudinal analysis of the humoral response to SARS-CoV-2 spike RBD in convalescent plasma donors

Hema-Quebec, the blood supplier in the Province of Quebec, Canada, collects and tests convalescent plasma used in a clinical trial to determine the clinical efficacy of this product for the treatment of hospitalized COVID-19 patients. So far, we have collected 1159 plasma units from 282 COVID-19 convalescent donors. The presence of antibodies to the receptor binding domain (RBD) of SARS-CoV-2 spike protein in convalescent donors was established at the first donation. Seropositive donors were asked to donate additional plasma units every six days. Until now, 15 donors have donated at least four times and, in some cases, up to nine times. This allowed us to perform a longitudinal analysis of the persistence of SARS-CoV-2 RBD-specific antibodies in these repeat donors, with the first donation occurring 33-77 days after symptoms onset and donations up to 71-114 days after symptoms onset thereafter. In all donors, the level of antibodies remained relatively stable up to about 76 days after symptoms onset but then started to decrease more rapidly to reach, in some convalescent donors, a seronegative status within 100-110 days after symptoms onset. The decline in anti-RBD antibodies was not related to the number of donations but strongly correlated with the numbers of days after symptoms onset (r = 0.821). This suggests that de novo secretion of SARS-CoV-2 RBD antibodies by short-lived plasma cells stopped about 2-3 months after disease onset, an observation that has important implications for convalescent plasma collection and seroprevalence studies undertaken several months after the peak of infection.

immunology↗
bioRxiv2020

Helium As an In Vitro Furin Inhibitor

AimsCertain cancers, pathogenic infections, and other diseases are facilitated by the host enzyme furin, a calcium-dependent serine protease that is the most prominent member of the family of proprotein convertases. Furin and the other proprotein convertases modify certain other proteins to change them from their inactive to active forms. Previous attempts to find an effective, non-toxic furin inhibitor to treat diseases facilitated by furin have had only limited success, due to toxicity or large molecular size that impedes absorption of the molecule. This has placed increased importance on the development of small-molecule furin inhibitors. The object of this study was to consider the effect of the noble gas helium as a furin inhibitor. MethodsThis study uses a fluorometric furin inhibition assay to compare the enzymatic activity of recombinant human furin after exposure to balloon-grade helium gas, compared to the enzymatic activity of untreated recombinant human furin. ResultsHelium exposure was found to decrease the in vitro enzymatic activity of recombinant human furin by as much as 55%; this inhibition lasted for up to 16 hours. Fluorescence measurements of enzymatic activity were taken for 24 hours. ConclusionsThese findings appear to be the first to report helium as a partial furin inhibitor. The observed partial inhibition was consistent throughout the experiment. The effectiveness of helium as a partial furin inhibitor, its favorable side-effect profile, and its long history of safe use in respiratory therapy, when mixed with 20% oxygen or more and administered under direct medical supervision, make it a promising treatment for diseases facilitated by furin or its substrates. Further studies in cell culture or clinical trials may expand its clinical role for such diseases.

pharmacology and toxicology↗
bioRxiv2021

Development and characterization of a high fat diet-streptozotocin induced type 2 diabetes model in nude athymic rats

People with diabetes mellitus (DM) are at an increased risk for myocardial infarction (MI) than age matched people without DM. However, assays for pre-clinical therapy are performed in animal models of ischemia that lack the co-morbid conditions present in patients with MI, such as DM. This contributes to the failure to translate pre-clinical trials results to the clinic. Thus, to increase the clinical relevance of xenograft studies in pre-clinical models, it is important to have a DM model in animals that are immunodeficient. Here, we developed a type 2 diabetes mellitus (T2D) model in nude athymic rats using high-fat diet and streptozotocin (HFD-STZ). Nude athymic rats were randomized into a control group (normal chow) or a HFD (45% fat, 20% protein and 35% carbohydrate)-STZ group. STZ (35 mg/kg i.v.) or vehicle was administered 8 weeks after HFD feeding started. Assessments were done longitudinally and at week 9 (endpoint). The HFD-STZ group showed mild hyperglycemia pre STZ administration (7.7 {+/-} 0.3 mM vs 5.8 {+/-} 0.2 mM in control) by week 8. In addition, plasma insulin levels were increased and the HOMA index was 2.5-times higher in the HFD-STZ. The HFD-STZ group showed increased fasting (147%) triglycerides. After STZ-administration, blood glucose levels increased substantially (23.6 {+/-} 1.4 mM vs 5.5 {+/-} 0.3 mM in control). The HFD-STZ treated animals also showed increased left ventricular wall thickness, cardiac hypertrophy and fibrosis, reduced cardiac function compared to normal chow control. In line with the HFD-STZ model in immunocompetent rats, the HFD-STZ treatment of athymic rats recapitulates key features of T2D, including aspects of established clinical diabetic cardiomyopathy and should be suitable for xenograft studies in the context of T2D.

pathology↗
bioRxiv2025

Engineering nanoparticles that target fibroblast activation protein in cardiac fibrosis

Cardiac fibrosis and dysfunction, hallmarks of debilitating heart disease, are driven by fibroblast activation protein alpha (FAP). The specificity of FAP to the disease creates an opportunity for directed drug delivery, as FAP delineates the fibrotic region. We leverage this vulnerability to target the fibrotic region of the heart, using anti-FAP antibody-modified nanoparticles (NPs) that encapsulate and release the highly specific FAP inhibitor, talabostat. NP crosslinking with an FAP-sensitive peptide attenuates passive release, which is then accelerated in the presence of FAP. Intravenous administration of these NPs results in reversal of established cardiac fibrosis and dysfunction in a rat model of myocardial infarction, using a talabostat dose that is 400,000-fold lower than that used in clinical trials. This innovative and clinically translatable strategy enables targeted drug delivery, overcoming limitations of systemic approaches by reducing therapeutic dose, minimizing off-target effects, and accommodating patient-specific variability in FAP expression.

bioengineering↗
bioRxiv2019

PK-DB: PharmacoKinetics DataBase for Individualized and Stratified Computational Modeling

A multitude of pharmacokinetics studies have been published. However, due to the lack of an open database, pharmacokinetics data, as well as the corresponding meta-information, have been difficult to access. We present PK-DB (https://pk-db.com), an open database for pharmacokinetics information from clinical trials including pre-clinical research. PK-DB provides curated information on (i) characteristics of studied patient cohorts and subjects (e.g. age, bodyweight, smoking status); (ii) applied interventions (e.g. dosing, substance, route of application); (iii) measured pharmacokinetic time-courses; (iv) pharmacokinetic parameters (e.g. clearance, half-life, area under the curve). Key features are the representation of experimental errors, the normalization of measurement units, annotation of information to biological ontologies, calculation of pharmacokinetic parameters from concentration-time profiles, a workflow for collaborative data curation, strong validation rules on the data, computational access via a REST API as well as human access via a web interface. PK-DB enables meta-analysis based on data from multiple studies and data integration with computational models. A special focus lies on meta-data relevant for individualized and stratified computational modeling with methods like physiologically based pharmacokinetic (PBPK), pharmacokinetic/pharmacodynamic (PK/DB), or population pharmacokinetic (pop PK) modeling.

bioinformatics↗
bioRxiv2019

Epigenetic regulation of protein translation in KMT2A-rearranged AML

Inhibition of the histone methyl-transferase DOT1L (KMT4) has shown encouraging activity in preclinical models of KMT2A (MLL)-rearranged leukemia. The DOT1L inhibitor pinometostat (EPZ5676) was well tolerated in early phase clinical trials and showed modest clinical activity, including occasional complete responses (CRs) as single agent. These studies support the development of combinatorial therapies for KMT2A-rearranged leukemias. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination of a DOT1L inhibitor with the protein synthesis inhibitor homoharringtonine (HHR).\n\nEZH2 is the catalytic histone methyltransferase in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has reported preclinical activity in KMT2A-rearranged leukemia. We found that the H3K79 and H3K27 methyl marks are not dependent on each other, and that DOT1L and EZH2 inhibition affect largely distinct gene expression programs. In particular, the KMT2A/DOT1L target HOXA9, which is commonly de-repressed as a consequence of PRC2 loss or inhibition in other contexts, was not re-activated upon dual DOT1L/EZH2 knockout or inhibition. Despite encouraging data in murine KMT2A-MLLT3 transformed cells suggesting synergy between DOT1L and EZH2 inhibition, we found both synergistic and antagonistic effects on a panel of human KMT2A rearranged cell lines. Combinatorial inhibition of DOT1L and EZH2 is thus not a promising strategy. We identified opposing effects on ribosomal gene transcription and protein translation by DOT1L and EZH2 as a mechanism that is partially responsible for observed antagonistic effects. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (HHR), supporting further preclinical/clinical development of this combination.

cancer biology↗
bioRxiv2026

Biomarker Variability Limits Individualized Amyloid Time Estimation in Alzheimer Disease

ObjectiveDisease progression modeling (DPM) or "amyloid time" is increasingly used to stage Alzheimer disease (AD). DPM performance depends on within-individual heterogeneity in rates of pathological accumulation as well as test-retest reliability of the biomarker. The relative contributions of these variabilities have not been systematically assessed. This would be particularly relevant if extrapolations from DPM were to be used to make individual-level predictions for research, clinical trials, or potentially future clinical practice. MethodsWe conducted simulation studies incorporating empirically-derived noise properties from amyloid biomarkers to assess the contributions of inter- and intra-individual variability. Findings generalized in an autosomal dominant AD cohort with amyloid positron emission tomography (PET), cerebrospinal fluid (CSF), and plasma biomarkers and in a sporadic AD cohort with both amyloid PET and plasma biomarkers. We assessed group level DPM performance via mean average error (MAE) and root mean squared error (RMSE). At the individual level, we evaluated distinctness of distributions of biomarker levels associated with specific disease timings. ResultsInter-individual variability was the dominant source of error in temporal estimates. Intra-individual variability reduced estimate stability. Optimal performance occurred in biomarkers with positive average accumulation rates where a subset of individuals had exceptionally high levels of accumulation. In research study data, amyloid PET outperformed CSF and plasma biomarkers. InterpretationDPM is fundamentally constrained by dynamic range, variability, and test-retest reliability of the biomarker of interest. Current DPM approaches are more robust at the group level, particularly when applied to biomarkers with more than 10-15% variability like fluid biomarkers. FundingNational Institute on Aging, Alzheimers Association, German Center for Neurodegenerative Diseases, Raul Carrea Institute for Neurological Research, Japan Agency for Medical Research and Development, Korean Ministry of Health & Welfare and Ministry of Science and ICT, Spanish Institute of Health.

bioinformatics↗