Biology subjects

Arora, S.

Publications and source records attributed to Arora, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

SynGlue: AI-Driven Designer for Clinically Actionable Multi-Target Therapeutics

The rational design of protein degraders, such as proteolysis-targeting chimeras (PROTACs), requires the simultaneous optimization of multiple molecular properties, a complex challenge that limits efficient discovery. Here, we introduce SynGlue, a generative artificial intelligence (AI) framework that addresses this challenge through two core modules: data-driven, leveraging large-scale protein-ligand intelligence, and structure-guided, for physics-aware molecular design. SynGlue harness MagnetDB, a curated database of 6.37 million experimental protein-ligand interactions, and couples it with deep learning models that quantitatively predict degradation potency (DC50), maximal degradation (Dmax), and guide ternary-complex-compatible linker design. Benchmarked against 6,935 compounds, SynGlue demonstrates superior performance in relevant pharmacology prediction. To validate SynGlue, we engineered degraders for BRD4 and GSPT1. Our data-driven design for BRD4 yielded compounds with novel warhead scaffolds (<50% warhead similarity with known PROTACs), which proved to be potent degraders in vitro (DC50 = 0.19 nM) and efficacious in vivo in mouse models. Independently, our structure-guided de novo design for GSPT1 produced ultrapotent degraders (DC50 {approx} 0.0011 M) that are also effective both in vitro and in vivo, uncovering a new oncogenic dependency. By unifying data-driven and physics-aware design, SynGlue establishes a generalizable AI framework for the rapid development of clinically relevant protein degraders, with principled extension to other multi-target modalities.

bioinformatics↗

bioRxiv2025

MutAIverse: An AI-Powered, Mechanism-backed Platform for Discovering Novel DNA Adducts and their precursor Genotoxins

Genotoxin exposure leads to DNA adduct formation, potentially causing mutations if unrepaired. Current DNA adductomics platforms or analytical workflows are limited by incomplete spectral libraries, reliance on experimentally validated adducts, limited cellular contexts, and inefficient computational methodologies. We introduce MutAIverse, an advanced AI-driven DNA adductomics analysis platform that overcomes these limitations by leveraging intracellular mechanistic modeling of genotoxin bioactivation to construct a comprehensive DNA adduct library and offers advanced spectral mapping. MutAIverse integrates experimentally validated and chemically valid putative DNA adducts, enabling interpretable retrograde tracking to parental genotoxins. Validation of MutAIverse against experimental MS/MS datasets demonstrated the detection of both known and novel adducts. Furthermore, application to in-house generated DNA adductomics data from tissue biopsies of smokeless tobacco-induced head and neck cancer patients revealed selective enrichment of both novel and known DNA adducts; a subset of them was validated using MS/MS analysis. Collectively, MutAIverse provides a robust, end-to-end, and interpretable platform for advanced DNA adductomics analysis.

bioinformatics↗

bioRxiv2025

Orai3 orchestrates gemcitabine resistance in pancreatic cancer via NFATc1-SLIT3 axis

Pancreatic Cancer (PC) is one of the most aggressive cancers and is associated with poor prognosis. Gemcitabine is the first-line chemotherapy for PC. While gemcitabine-based regimes offer survival benefits, the acquired gemcitabine resistance leads to reoccurrence, metastasis and the long-term survival rate remains dismal. Although there is substantial clinical evidence for gemcitabine resistance, the cellular and molecular mechanisms driving gemcitabine resistance remain largely unappreciated. Here, we reveal that Orai3, a Ca2+ selective channel, is a crucial driver of gemcitabine resistance. We demonstrate that Orai3 is upregulated in gemcitabine-resistant PC cells. Orai3 silencing in these cells decreases proliferation, induces cell cycle arrest, enhances apoptosis, and moderates stemness characteristics. Notably, studies in zebrafish model corroborate the significance of Orai3 in gemcitabine resistance in vivo. Mechanistically, our unbiased RNA-seq analysis coupled with robust functional studies show that SLIT3 works downstream of Orai3 to drive gemcitabine resistance. Finally, we report that NFATc1 transcription factor bridges Orai3 to SLIT3 transcription. Taken together, this study identifies Orai3 as a key orchestrator of gemcitabine resistance and uncovers a unique Orai3-NFATc1-SLIT3 signaling module that drives chemoresistance. Hence, this work reveals Orai3 as a promising target for synergistic therapeutic approach to combat chemoresistance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/671673v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@16fea2org.highwire.dtl.DTLVardef@1a77a3forg.highwire.dtl.DTLVardef@4aef28org.highwire.dtl.DTLVardef@19b62a3_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights{Rightarrow} Orai3 is upregulated in gemcitabine-resistant pancreatic cancer cells {Rightarrow}Orai3 is essential for chemo-sensitivity, migration, invasion and stemness in vitro {Rightarrow}Orai3 regulates metastasis of gemcitabine-resistant pancreatic cancer cells in vivo {Rightarrow}Unbiased RNA-seq reveals that SLIT3 functions downstream of Orai3 to drive gemcitabine resistance {Rightarrow}NFATc1 transcription factor bridges Orai3 to SLIT3 transcription

cancer biology↗

bioRxiv2025

The Tricuspid Valve is Transcriptionally Active During Prolonged Pressure Overload, Right-Sided Heart Failure, and Valve Regurgitation

BackgroundRight-sided heart failure (RHF), in the presence of tricuspid valve regurgitation (TR), can result from left-sided heart failure (LHF), pulmonary hypertension (PH), or heart malformations. The occurrence of RHF and TR represents a critical indicator of hospitalization rates and all-cause mortality. However, RHF has remained understudied, specifically with respect to the tricuspid valve, with few animal models to investigate the transformative processes and identify novel interventions. MethodsUsing the outbred sheep (Ovis aries) model of pulmonary artery banding (PAB) that induces RHF and TR, we generated three batches of ribosomal reduced RNA sequencing for 354 samples (NCBI SRA PRJNA1182691) containing right ventricle, left ventricle, each tricuspid valve leaflet, each mitral valve leaflet, and the pulmonary artery that represents both male and female sheep. The reads were assembled into a de novo sheep heart transcriptome for differential analysis. ResultsThe de novo sheep heart transcriptome enhanced transcript mapping of reads by 43-45% in the heart valves relative to the known sheep reference transcriptome. The identified transcripts produce validated tissue-specific pathways in ventricles (2,756 isoforms), pulmonary arteries (535 isoforms), and valves (1,215 isoforms), with transcript differences between the mitral and tricuspid valve involved in extracellular and endocrine signaling. The transcriptome also produced robust sex differences encoded by sex chromosomes and autosomes, highlighting epigenetic and sex hormone differences in the heart. Echocardiography and differential expression suggest that 8 weeks after PAB, the right ventricle has extensive morphological changes and known stress-induced lipid processing dysregulation. At 16-weeks post-PAB, tricuspid valve leaflets show the most significant transcriptional changes, with alterations in endocrine and immune pathway genes involved in cellular and extracellular remodeling. Genes within the tricuspid valve with differential expression and known human or mouse heart phenotypes include FLNA, LTBP4, VDR, CR2, PIGQ, CENPF, ACKR3, CR1, KLF2, and HIF3A. ConclusionsThis project highlights the complexity of heart valve tissues and their transcriptional activity in a sheep model of RHF. It suggests potential therapeutic interventions in heart valve remodeling in PAH, RHF, and TR. This work highlights the need for further human and model organism research into the dynamic valve cells and genes. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABS- Improved cardiac and valve specific transcriptome mapping through de novo transcriptome for clinically relevant ovine model. - Tricuspid valves show a sex dependent and active transcriptional response to pulmonary artery banding induced pulmonary hypertension and right sided heart failure. - Transcriptional phenotypes in ovine model mirror known human heart disease phenotypes. - Several transcripts identified with therapeutic potential for treating pressure overload conditions such as pulmonary hypertension. What Are the Clinical Implications?- Tricuspid valve remodeling due to pulmonary hypertension is accompanied by transcriptional alterations, and mechanical alterations alone may not be sufficient to address valve insufficiency. - Valve and ventricle sex specific gene expression changes following pulmonary hypertension indicate a potential role for hormonal influences and a need for personalized treatment strategies. - Improved patient outcomes for right heart failure, including diagnosis, early detection, and improved treatment strategies can be elucidated through the ovine model and supported through expanded interrogation of human tissues.

genomics↗

bioRxiv2025

Affordable plasmonic biosensing: democratizing SERS with scalable, field-compatible substrate fabrication

Efficient and accurate plasmonic biosensing in the field remains a significant challenge. Despite its potential to revolutionize point-of-care (PoC) diagnostics through its unparalleled sensitivity and precise molecular fingerprinting capabilities, adoption of traditional surface-enhanced Raman spectroscopy (SERS) in the field has not proven feasible yet. High production and material costs, complex fabrication methods, reliance on specialized equipment, and persistent issues with sensor stability and reproducibility continue to impede development for PoC use. Addressing these challenges, this study innovates a democratized and cost-effective fabrication kit that enables the production of SERS substrates using commonly available materials and straightforward electrochemistry techniques without compromising on sensitivity and reproducibility. Importantly, this method leverages commercially available bottled water and simple battery-powered fabrication, thereby eliminating reliance on a power grid and enabling the local production of biosensors in resource-restricted and conflict-affected areas. The cost of producing the fabrication kit is $39.54 with raw materials purchased at bulk retail prices, while the cost of consumables for fabricating each test is just 1.33{cents}. To ensure real-world feasibility, we conducted a comprehensive reproducibility analysis, where consistent plasmonic enhancement was observed across multiple production batches. Furthermore, we demonstrated their efficacy in two critical applications: the rapid detection of bacteria and pesticides. We detect trace levels of pesticides such as Thiram and Thiabendazole down to 0.1 ppm using a digital SERS approach. We also demonstrated the identification of bacteria isolated from culture, namely Escherichia coli and Bacillus subtilis. We envision that this label-free, high-sensitivity substrate, when paired with portable Raman spectrometers, could open doors for a new era of field-deployable biosensing, paving the way for its adoption for a plethora of applications, from public health to food testing.

bioengineering↗

bioRxiv2025

Targeted chelation therapy decreases NLRP3 expression by vascular cells and acts as senomorphic in Chronic Kidney Disorder induced Vascular Calcification.

BackgroundChronically high levels of phosphate (P) in the serum caused by chronic kidney disease (CKD) induce osteogenic changes in the aortic Vascular Smooth Muscle Cells (VSMCs). Premature onset of cellular senescence is observed in these phenotypically transitioned cells, which plays a critical role in pathology of vascular calcification. We have previously shown that EDTA therapy can remove calcium deposits from the arteries in a rat model of CKD and reduces the expression of osteogenic markers in the aorta. In the current study we evaluated if chelation therapy with EDTA has senotherapeutic potential and could also decrease the accumulation of senescent cells in the aorta once it has calcified. MethodsWe used an adenine diet-based rodent model of late-stage CKD and an ex-vivo aortic ring culture model to evaluate the senotheraputic potential of EDTA loaded-human serum albumin nanoparticles tagged with anti-elastin antibody-Flexibzumab (EDTA-NP). For validation we performed a comparative proteomics analysis on the total proteins harvested from the abdominal aortas of the EDTA nanoparticle treated and untreated animals. ResultsOur results show that targeted chelation therapy with EDTA-NP decreases the percentage of SA-beta gal positive senescent cells in the calcified aorta and acts as senomorphic by decreasing NLRP3 inflammasome formation which is a major intracellular source of Senescence associated secretory phenotype (SASP). ConclusionFor the first time, the current study provides a proof of concept on the senotheraputic potential of a targeted chelation therapy and its capacity to modulate SASP from the senescent cells accumulates in calcified aorta. HighlightsO_LIOur findings show that chelation therapy can act as senomorphic, and increases the life span of rodents suffering from heavy vascular calcification. C_LIO_LIChelation therapy decreases senescent cell accumulation, SASP and NLRP3 expression in the aorta. C_LIO_LIChelation therapy is a novel method for reprogramming senescent cells in the aorta to prevent their phenotypic switching to inflammatory senescent cells and ultimately to osteoblasts. C_LIO_LICurrent data have provided a new hypothesis that agents that restore mineral imbalance in the cellular microenvironment (in this case, EDTA) have the potential to act as senomorphics, which can serve as safer therapeutic alternatives over senolytics to treat vascular calcification by decreasing apoptosis. C_LI

pharmacology and toxicology↗

bioRxiv2025

Designed NGF mimetics with reduced nociceptive signatures in neurons

The clinical use of Nerve Growth Factor (NGF) for neuronal regeneration has been hampered by pain sensitization side effects. NGF signals through the receptor tyrosine kinase TrkA and the co-receptor p75NTR; pain sensitization is thought to involve p75NTR. We sought to overcome this limitation by de novo design of a TrkA agonist that does not bind p75NTR. We designed homodimeric TrkA engaging constructs that dimerize TrkA subunits in a variety of geometries, and identified those eliciting the strongest signaling. The resulting designed agonists are able to stimulate transdifferentiated neurons and neuroblastoma cell lines, leading to neurite outgrowth and neuronal differentiation, with considerably reduced transcription of inflammation and pain related genes. These agonists are promising candidates for promoting neuronal regeneration without adverse side effects. HighlightsO_LIDe novo designed TrkA agonists activate MAPK and PI3K-AKT signaling C_LIO_LIRigid fusions allow for highly tunable signaling signatures C_LIO_LITrkA agonists lead to neurite outgrowth in neuroblastoma cells comparable to retinoic acid C_LIO_LIModulation of the TrkA pathway without co-stimulating p75NTR leads to a downregulation of inflammatory and nociceptive signature in neurons. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/648806v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c48066org.highwire.dtl.DTLVardef@c9c5a7org.highwire.dtl.DTLVardef@cf8ebdorg.highwire.dtl.DTLVardef@a4345a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Addressing efficacy of everyday hygiene cleansing products in context of sustainable handwashing behavior in the post-pandemic era

Simple hygiene behaviour such as washing hands is key to improving health of individuals and reducing community transmission of communicable diseases such as respiratory and enteric infections. Consistent and relentless messaging by global and local health authorities had resulted in heightened hygiene awareness amongst the public during the COVID-19 pandemic. Unfortunately, many of the hygiene behaviours practiced during the pandemic have proven to be unsustainable in the immediate period following the pandemic. While CDC guidelines suggest washing hands with soap for a minimum of 20 seconds to prevent the spread of germs, a hygiene interventions effectiveness must be evaluated in context of the prevalent hygiene behaviour. Here we report our findings from an observational study conducted across India, Pakistan, Saudi Arabia, and United Arab Emirates focusing on handwashing habit of individuals in the post-pandemic period. Across all geographies, the time spent for lathering product on skin (contact time) for a significant majority of individuals recruited for the study was found to be 10 seconds and less. To ensure that marketed hygiene formulations such as liquid cleansers and sanitizers are efficacious in inactivating pathogens under conditions practiced by the majority as a part of their daily hand washing practices, we have investigated the in-vitro antimicrobial efficacy of several hygiene cleansing formulations at 10 seconds of contact time. Our results show that well-formulated cleansing solutions can reduce the input titre of both bacterial and viral pathogens by 99.9% or more, even with brief contact. With the global resurgence of both existing and emerging pathogens in the post-pandemic world, promoting sustainable handwashing practices for infection prevention remains a challenge as it is deeply rooted in the socio-economic fabric of many countries. Therefore, it is crucial to conduct regular studies like this to reassure end-users and combat complacency regarding the composition of everyday hygiene products. HighlightsO_LIStudies of handwashing behavior in 4 countries reveals significantly short lathering duration C_LIO_LIAntimicrobial efficacy testing of marketed products was conducted against a range of pathogens C_LIO_LISuitably formulated hygiene products can inactivate pathogens at short durations of contact C_LI

microbiology↗

bioRxiv2025

Evaluation of Antioxidant Anticancer and Antimicrobial Activities of Datura Leaf Extracts: A Microbiological Perspective

Datura, a genus of flowering plants, has long been used in traditional medicine owing to its pharmacological properties. This study investigated the antioxidant and antimicrobial activities of Datura leaf extracts by using in vitro assays. Antioxidant potential was evaluated using standard methods, while antimicrobial activity was assessed against various bacterial and fungal strains using the disk diffusion method. The results demonstrated significant antioxidant properties and broad-spectrum antimicrobial activity, supporting the potential of Datura as a source of bioactive compounds for pharmaceutical applications. The findings of this study elucidate the potential of datura leaf extracts as natural alternatives to synthetic antioxidants and antimicrobial agents in various industries. Further research is warranted to explore the specific bioactive compounds responsible for these properties and to investigate their mechanisms of action. Additionally, future studies should focus on optimizing extraction methods to enhance the potency and efficacy of Datura-derived products for potential therapeutic applications.

bioengineering↗

bioRxiv2025

Systems Pharmacology Screening Reveals Mertk and other Targetable Kinases in Supratentorial Ependymomas

Ependymomas (EPN) are rare central nervous system tumors that account for approximately 10% of intracranial tumors in children and 4% in adults. Despite their clinical and molecular heterogeneity, spanning supratentorial, posterior fossa, and spinal subtypes, treatment remains limited to surgery and radiotherapy, with chemotherapy offering minimal benefit. Here, we performed transcriptomic analysis of 370 human ependymoma samples and identified two distinct molecular subgroups: EPN-E1 and EPN-E2. The EPN-E1 cluster is enriched for supratentorial tumors harboring ZFTA-RELA fusions (ZFTA-RELAfus), which occur in over 70% of cases and are associated with poor prognosis. To identify targeted therapies for this aggressive subtype, we validated a ZFTA-RELAfus mouse model that recapitulates the human EPN-E1 transcriptome and used it for target discovery. Through Kinome Regularization, a machine learning-driven polypharmacology approach, we identified MERTK as a critical regulator of tumor cell viability. Genetic depletion or pharmacologic inhibition of Mertk reduced cell growth ex vivo, and treatment with a clinical-grade MERTK inhibitor significantly suppressed tumor proliferation in vivo. Both human EPN-E1 tumors and ZFTA-RELAfus mouse tumors exhibited elevated expression of MERTK and its ligand GAS6, and MERTK inhibition led to suppression of pro-survival signaling pathways including MEK/ERK and PI3K/AKT. Notably, over 80% of genes upregulated in ZFTA-RELAfus tumors were downregulated following MERTK inhibition, indicating a strong dependency on this pathway for tumor maintenance. These findings define a signaling vulnerability in ZFTA-RELA-driven ependymomas and support the clinical development of MERTK-targeted therapies for patients with the high-risk EPN-E1 subtype.

cancer biology↗

bioRxiv2025

Pea-Saving Partners: Bacillus and Pseudomonas combat downy mildew in pea crops

Downy mildew (DM) is a destructive disease that significantly reduces the yield and quality of important pulses (legumes) and horticultural crops, particularly during humid and cool seasons. This disease is caused by obligate and host-specific oomycete pathogens. Controlling the pathogen is challenging due to its long-term survival as spores and its rapid mutation. Use of chemical pesticides has been the most effective method to control DM pathogens, but their environmental hazards are a global concern. Current research is focused on exploring the potential of microbial biological control agents (MBCA), particularly rhizobacteria strains of the genera Bacillus and Pseudomonas, which have shown suppression of plant pathogens. However, to date, no MBCA has been reported to be effective against DM pathogens in pulses. We investigated the effectiveness of Bacillus and Pseudomonas strains as potential biopesticides against the pea downy mildew pathogen Peronospora viciae f. sp. pisi (Pvp). In our study, in vitro bioassays showed 100% inhibition of Pvp spore germination compared to the control. In planta antagonism assays further demonstrated significant suppression (>80%) of Pvp sporulation in pea plants sprayed with strains of Bacillus velezensis or P. fluorescens or their filtrates. The drench application also showed significant effects where either a Pseudomonas or cold-adapted Bacillus strain was used. We observed a synergistic effect for the dual foliar application of the microbes compared to individual application (27.6 to 46.7% suppression). Furthermore, the results from the molecular biomass analysis were consistent with the results of the sporulation assays. This demonstrates the strong interactive and promotive benefits of using Bacillus and Pseudomonas as biocontrol agents Based on these results, we conclude that these MBCAs could be effective in combatting Pvp infections in the field.

plant biology↗

bioRxiv2024

Transcriptomic landscape identifies two unrecognized ependymoma subtypes and novel pathways in medulloblastoma

Medulloblastoma and ependymoma are common pediatric central nervous system tumors with significant molecular and clinical heterogeneity. We collected bulk RNA sequencing data from 888 medulloblastoma and 370 ependymoma tumors to establish a comprehensive reference landscape. Following rigorous batch effect correction, normalization, and dimensionality reduction, we constructed a unified landscape to explore gene expression, signaling pathways, RNA fusions, and copy number variations. Our analysis revealed distinct clustering patterns, including two primary ependymoma compartments, EPN-E1 and EPN-E2, each with specific RNA fusions and molecular signatures. In medulloblastoma, we observed precise stratification of Group 3/4 tumors by subtype and in SHH tumors by patient age. This landscape serves as a vital resource for identifying biomarkers, refining diagnoses, and enables the mapping of new patients bulk RNA-seq data onto the reference framework to predict biology and outcome from nearest neighbor analysis facilitate accurate disease subtype identification. The landscape is accessible via Oncoscape, an interactive platform, empowering global exploration and application. One Sentence SummaryA landscape built using only Transcriptomic analysis for medulloblastoma and ependymoma reveals novel insights about subtype-specific biology.

bioinformatics↗

bioRxiv2024

Age-induced BMP signaling inhibits the ICAT-mediated Wnt pathway to promote osteoarthritis.

Osteoarthritis (OA) is a highly prevalent and debilitating musculoskeletal disorder that affects billions of aging individuals worldwide, causing chronic pain, impaired mobility, and a substantial decline in quality of life, yet effective disease-modifying therapies remain unavailable. Moreover, existing experimental models, however, inadequately capture the complex, age-related factors that contribute to OA pathogenesis, limiting our understanding of underlying molecular mechanisms and hindering therapeutic development. To address this gap, we examined mouse articular cartilage (AC) during natural aging, without surgical or chemical intervention. In young AC, BMP ligand expression is restricted to the bone-cartilage junction, limiting BMP signaling despite widespread BMPR1A receptor expression, while Wnt/{beta}-catenin signaling predominates in the outer layers. With aging, BMP ligand expression expands throughout the cartilage, leading to widespread activation of BMP signaling, which induces ICAT expression, a known inhibitor of Wnt/{beta}-catenin signaling. This elevated BMP signaling triggers chondrocyte hypertrophy while concurrently suppressing Wnt/{beta}-catenin activity. Moreover, genetic or pharmacological activation of BMP signaling in young AC recapitulates the cellular and molecular features of aged cartilage, independent of chronological age. Collectively, our findings demonstrate that an age-dependent shift in BMP-Wnt signaling disrupts AC homeostasis and drives OA progression in mice. Author SummaryDespite the prevalence of osteoarthritis in aging individuals, there are no effective treatments that can stop or reverse disease progression. In this study, we investigated how joint cartilage changes during natural aging in mice. We observed that healthy, young articular cartilage exhibits robust Wnt/{beta}-catenin with minimal BMP siganling. With age, this balance shifts, with BMP signaling becoming dominant, coupled with reduced Wnt/{beta}-catenin activity. This disruption of Wnt-BMP homeostasis induces the pathogenesis of OA. Importantly, local inhibition of BMP signaling significantly alleviates the severity of the disease. Overall, these results show that age-associated changes in key signaling pathways drive OA progression and suggest potential targets for therapeutic intervention. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/616036v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@b2be12org.highwire.dtl.DTLVardef@dcb1f3org.highwire.dtl.DTLVardef@126fc40org.highwire.dtl.DTLVardef@7af96c_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2024

CRISPR screen for rAAV production implicates genes associated with infection

Recombinant adeno-associated virus (rAAV) vectors are an effective and well-established tool in the growing gene therapy field, with five FDA-approved AAV-mediated gene therapies already on the market and numerous more in clinical trials. However, manufacturing rAAV vectors is an expensive, timely, and labor-intensive process that limits the commercial use of AAV-mediated gene therapies. To address this limitation, we screened producer cells for genes that could be targeted to increase rAAV yield. Specifically, we performed a CRISPR-based genome-wide knockout screen in HEK 293 cells using an antibody specific to intact AAV2 capsids coupled with flow cytometry to identify genes that modulate rAAV production. We discovered that the knockout of a group of heparan sulfate biosynthesis genes previously implicated in rAAV infectivity decreased rAAV production. Additionally, we identified several vesicular trafficking proteins for which knockout in HEK 293 cells increased rAAV yields. Our findings provide evidence that host proteins associated with viral infection may have also been co-opted for viral assembly and that the genetic makeup of viral producer cells can be manipulated to increase particle yield.

cell biology↗

bioRxiv2024

LncRNA TAAL is a Modulator of Tie1-Mediated Vascular Function in Diabetic Retinopathy

Diabetic retinopathy (DR), a leading cause of vision impairment and blindness, is characterized by abnormal retinal vascular changes due to chronic hyperglycemia. The Tie-1 signaling pathway, essential for vascular growth and remodeling, has emerged as a key therapeutic target, though its molecular mechanisms and interactome remain largely unclear. Through a protein-centric approach, we identified a novel lncRNA and named it Tie1-associated angiogenic lncRNA (TAAL). TAAL lncRNA regulates endothelial cell migration, proliferation, tube formation, and permeability by modulating ER-calcium homeostasis and cytoskeleton dynamics. In zebrafish, taal modulation led to angiogenic defects, which were rescued by human TAAL orthologue. Our molecular studies further revealed that TAAL negatively regulates Tie1 protein via ubiquitin-mediated degradation. Notably, TAAL expression is upregulated in the blood of DR patients and downregulated in endothelial DR cell models. Overexpression of TAAL restored endothelial permeability and VE-cadherin surface expression. These findings establish TAAL as a novel regulator of Tie1 protein turnover, with potential therapeutic implications for diabetic retinopathy.

genetics↗

bioRxiv2024

Aggressive high-grade NF2 mutant meningiomas downregulate oncogenic YAP signaling via the upregulation of VGLL4 and FAT3/4.

Meningiomas are the most common primary brain tumors in adults. Although generally benign, a subset of meningiomas is of higher grade, shows aggressive growth behavior and recurs even after multiple surgeries. Around half of all meningiomas harbor inactivating mutations in NF2. While benign low-grade NF2 mutant meningiomas exhibit few genetic events in addition to NF2 inactivation, aggressive high-grade NF2 mutant meningiomas frequently harbor a highly aberrant genome. We and others have previously shown that NF2 inactivation leads to YAP1 activation and that YAP1 acts as the pivotal oncogenic driver in benign NF2 mutant meningiomas. Using bulk and single-cell RNA-Seq data from a large cohort of human meningiomas, we show that aggressive NF2 mutant meningiomas harbor decreased levels YAP1 activity compared to their benign counterparts. Decreased expression levels of YAP target genes are significantly associated with an increased risk of recurrence. We then identify the increased expression of the YAP1 competitor VGLL4 as well as the YAP1 upstream regulators FAT3/4 as a potential mechanism for the downregulation of YAP activity in aggressive NF2 mutant meningiomas. High expression of these genes is significantly associated with an increased risk of recurrence. In vitro, overexpression of VGLL4 resulted in the downregulation of YAP activity in benign NF2 mutant meningioma cells, confirming the direct link between VGLL4 expression and decreased levels of YAP activity observed in aggressive NF2 mutant meningiomas. Our results shed new insight on the biology of benign and aggressive NF2 mutant meningiomas and may have important implications for the efficacy of therapies targeting oncogenic YAP1 activity in NF2 mutant meningiomas.

molecular biology↗

bioRxiv2024

Early life intestinal inflammation alters gut microbiome, impairing gut-brain communication and reproductive behavior in mice

Despite recent advances in understanding the connection between the gut microbiota and the adult brain, there remains a wide knowledge gap in how gut inflammation impacts brain development. We hypothesized that intestinal inflammation in early life would negatively affect neurodevelopment through dysregulation of microbiota communication to the brain. We therefore developed a novel pediatric chemical model of inflammatory bowel disease (IBD), an incurable condition affecting millions of people worldwide. IBD is characterized by chronic intestinal inflammation, and has comorbid symptoms of anxiety, depression and cognitive impairment. Significantly, 25% of patients with IBD are diagnosed during childhood, and the effect of chronic inflammation during this critical period of development is largely unknown. This study investigated the effects of early-life gut inflammation induced by DSS (dextran sulfate sodium) on a range of microbiota, endocrine, and behavioral outcomes, focusing on sex-specific impacts. DSS-treated mice exhibited increased intestinal inflammation, altered microbiota membership, and changes in microbiota-mediated circulating metabolites. The majority of behavioral measures were unaffected, with the exception of impaired mate-seeking behaviors in DSS-treated males. DSS-treated males also showed significantly smaller seminal vesicles, lower circulating androgens, and decreased intestinal hormone-activating enzyme activity. In the brain, microglia morphology was chronically altered with DSS treatment in a sex-specific manner. The results suggest that early-life gut inflammation causes changes in gut microbiota composition, affecting short-chain fatty acid (SCFA) producers and glucuronidase (GUS) activity, correlating with altered SCFA and androgen levels. The findings emphasize the developmental sensitivity to inflammation-induced changes in endocrine signalling and underscore long-lasting physiological and microbiome changes associated with juvenile IBD. HighlightsEarly-life gut inflammation produces sex-specific effects on i) microbiome, ii) sex hormones and iii) behaviour. Both sexes show disrupted gut bacterial members that regulate sex hormone levels. Male mice demonstrate deficits in mate seeking, which may be mediated by reduced androgen levels. Both male and female mice demonstrate shifts in hippocampal microglial morphology.

neuroscience↗

bioRxiv2024

Optimized AAV capsids for diseases of the basal ganglia show robust potency and distribution in adult nonhuman primates

Huntingtons disease and other disorders of the basal ganglia create challenges for biomolecule-based medicines given the poor accessibility of these deep brain structures following intracerebral or intravascular delivery. Additionally, for adeno-associated viruses (AAVs) intravascular delivery exposes peripheral tissues to the vast majority of the therapy, increasing the risk of immune responses and the quantity and associated cost of goods required for therapeutically relevant brain penetration levels. Here, we found that low dose, low volume delivery of unbiased AAV libraries into a focused brain region allowed recovery of novel capsids capable of broad access to key deep brain and cortical structures relevant for human therapies at doses orders of magnitude lower than used in current clinical trials. One such capsid, AAV-DB-3, provided transduction of up to 45% of medium spiny neurons in the adult NHP striatum, along with substantial transduction of relevant deep layer neurons in the cortex. Notably, AAV-DB-3 behaved similarly in mice as in NHPs and also potently transduced human neurons derived from induced pluripotent stem cells. Thus, AAV-DB-3 provides a unique AAV for network level brain gene therapies that translates up and down the evolutionary scale for preclinical studies and eventual clinical use.

neuroscience↗