Biology subjects

Patel, R.

Publications and source records attributed to Patel, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Non-viral vasculogenic reprogramming restores cognition and mitigates pathology in Alzheimer's disease

Alzheimers Disease (AD) is characterized by progressive cognitive decline associated with amyloid-beta (A{beta}) plaques, neurofibrillaiy tangles, inflammation, synaptic loss, and profuse neuronal death. Accumulating evidence demonstrates that cerebrovascular impairment precedes the emergence of neuropathological hallmarks, implicating vascular dysfunction as an early contributor to AD onset and progression. We investigated a non-viral strategy to generate pro-vasculogenic fibroblasts by transiently overexpressing Et{upsilon}2, Foxc2, and Flii (EFF) as a potential cell-based therapy for neurovascular deficits in AD. To assess therapeutic potential, FFF-primc[d] fibroblasts were injected into a mouse model of AD (3xTg-AD) and wild-type controls via the intracerebroventricular (ICV) route, followed by cognitive assessments and subsequent brain tissue analyses. Our findings demonstrate that FFF-primed fibroblasts acquire vasculogenic properties, enhance cerebral blood flow (CBF), and alleviate spatial memory deficits in 3xTg-AD mice. Moreover, transplanted FFF-primed fibroblasts exhibited long-term survival, integrated into the brain vasculature, and promoted cortical vascular remodeling in the AD brain. Notably, ICV deployment of these cells is also correlated with reduced cortical amyloid-beta load, suggesting potential therapeutic benefits in reducing AD pathology. Transcriptomic analysis identified the activation of genes involved in fatty acid oxidation, such as Ppar, known for its anti-amyloidogenic and anti-inflammatory effects. Collectively, these findings highlight non- viral, reprogramming-based vasculogenic cell therapy as a promising strategy for Alzheimers disease, capable of alleviating cognitive decline and addressing AD pathology across cellular and tissue scales.

neuroscience↗

bioRxiv2025

A tailored in vivo CRISPR screen identifies BAP1 as a potent tumor suppressor of soft tissue sarcoma

Undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas (STS) in adults. Despite decades of research, therapeutic advancements for STS, including UPS, have remained limited. The genetic complexity of UPS, characterized by the absence of recurrent driver oncogene mutations, has hindered the development of effective targeted therapies beyond conventional chemotherapy and immunotherapy. To address this challenge, we conducted a customized in vivo CRISPR/Cas9 screen in mice to systematically identify potential tumor suppressors involved in UPS development. Our screen revealed BRCA1-associated protein 1 (Bap1) as a potent tumor suppressor in STS. Using total RNA sequencing, multiplex immunohistochemistry, and flow cytometry, we found that Bap1-deficient mouse sarcomas exhibit significant immune suppression. Further analysis indicated that polo-like kinase 1 (Plk1) is essential for the survival of Bap1-deficient sarcomas. Treatment with volasertib, a Plk1 inhibitor, markedly inhibited tumor growth in both syngeneic and spontaneous mouse models of Bap1-loss sarcoma. In conclusion, our findings suggest that PLK1 inhibition, or combined with immunotherapy, may represent a promising targeted therapeutic strategy for tumors lacking BAP1.

cancer biology↗

bioRxiv2025

Distinct Myogenic Stages Recapitulate Transcriptomic Networks in COPD Cachexia

BackgroundCachexia is an extrapulmonary manifestation of chronic obstructive pulmonary disease (COPD) characterized by weight loss and muscle wasting. Transcriptomic analysis of skeletal muscle may provide insight to COPD-cachexia-relevant dysregulation, including at the mitochondrial level, where dysfunction is a hallmark of muscle wasting. As muscle biopsies are invasive and yield finite tissue, human muscle derived cultures (HMDCs) can expand limited biopsy material for studying skeletal muscle dysregulation. However, utility of such models depends on their recapitulation of bulk skeletal muscle transcriptomic signatures. To address this, we tested whether COPD and COPD-cachexia dysregulated transcriptional signatures in bulk skeletal muscle are preserved across stages of early differentiation. MethodsVastus lateralis biopsies were collected from 15 (7M/8F, 64{+/-}9 years) participants; COPD n=6, COPD cachexia n=4, and n=5 age-matched controls. Cachexia was defined as weight loss coupled with reduced muscle strength, fatigue, anorexia, low muscle mass and/or systemic inflammation. Satellite cells were isolated and differentiated into HDMCs (myoblasts, myocytes, and myotubes). Transcriptomics data was generated from bulk skeletal muscle and HDMCs. Differential expression analysis identified transcripts significantly dysregulated (p<0.05) in COPD and/or COPD-cachexia. Weighted gene co-expression network analysis (WGCNA) preformed at the whole transcriptome and mitochondrial transcriptome levels, identified modules of co-expressed genes. Modules were tested for correlation with clinical traits and preservation between bulk tissue and HDMCs (Z-summary >2). Gene set enrichment analysis was performed for all modules. Results660 genes were significantly differentially expressed between COPD and control bulk skeletal muscle. The top upregulated and downregulated genes were IL21R-AS1 (Log2-Fold-Change [L2FC]=5.3, p=4.2x10-5) and top downregulated POU5F1B (L2FC=-5.8, p=3.1x10-2), respectively. Among the 492 genes significantly differentially expressed between COPD and COPD-cachexia bulk skeletal muscle. The top upregulated and downregulated genes were LINC02274 (L2FC=5.6, p=3.6x10-5) and ZFY (L2FC=-6.1, p=9.7x10-3) respectively. Modules 1, 9, A, B, D and H correlated with cachexia-relevant traits, and Modules 3, 7, G, and I correlated most strongly with COPD severity. Most modules (1, 2, 4, 7, 8, 9, A, B, H) were preserved across all states; Modules D and G were preserved in myoblasts only, and Module I in myotubes only. Preserved modules were enriched for contractile, inflammatory, oxidative phosphorylation, and fatty acid metabolism pathways. ConclusionsCo-expression modules linked to COPD and COPD-cachexia in bulk skeletal muscle were broadly preserved across all differentiation stages, with myoblast and myotubes most completely recapitulating disease-relevant transcriptional signatures. These findings support HMDCs, as tractable in vitro models preserving contractile, inflammatory, and mitochondrial signatures of COPD-associated muscle dysfunction.

genetics↗

bioRxiv2025

Multiomic characterization, early detection, and therapeutic targeting of myeloid sarcoma

Myeloid sarcoma, an aggressive extramedullary subtype of acute myeloid leukemia (AML), occurs in [~]10% of patients, and has not yet been included in large-scale genomic studies. The critical biological changes that drive tumor evolution are unknown, its detection in asymptomatic patients remains a clinical challenge, and treatment options are limited as patients are often excluded from clinical trials, rendering it a neglected disease entity. Based on comprehensive multi-omic profiling, we demonstrate that myeloid sarcoma evolves from medullary AML with distinct sitespecific clonal evolution patterns. Additionally, we show that circulating tumor DNA sequencing can serve as a non-invasive method for molecular profiling of myeloid sarcoma, offering a novel avenue in molecular diagnostics. We characterize unique transcriptional profiles of myeloid sarcoma, reflecting immune evasion and adaptation to an extramedullary microenvironment. We provide evidence for a key role of RAS pathway activation and demonstrate in murine models of myeloid sarcoma that RAS inhibition effectively reduces tumor burden. Overall, our data highlight key differences between medullary AML and myeloid sarcoma including universal molecular evolution and RAS pathway activation as hallmarks of the disease and nominate RAS inhibition as a promising therapeutic strategy for patients with myeloid sarcoma.

cancer biology↗

bioRxiv2025

Developing a nuclear transplantation model for Australian amphibian conservation using a common species of Australian Frog (Limnodynastes peronii)

As amphibian populations continue to decline, there is a need to establish procedures to store and recover genetic diversity. Nuclear transplantation (NT) is a potentially useful reproductive technology with applications to conservation in specific circumstances. Although sperm have been successfully cryopreserved and used in in vitro fertilization to produce sexually mature offspring, there are many endangered and even extinct amphibian species that only exist as stored frozen adult or larval tissues. The only way to recover that genetic diversity is through NT. Nuclear transplantation can introduce new genes into captive breeding programs from individuals where no viable gametes from that individual have been cryopreserved. Amphibian NT research was common during the middle to late 20th century; however, it was never conducted for amphibian conservation. Early amphibian NT experiments were conducted for the purpose of determining whether differentiation was a terminal process that involved the loss of reprogramming genes. However, following the success of mammalian NT, amphibian NT research declined, and the field has been at a standstill for decades. This study developed the first nuclear transplantation protocol for a native Australia frog using Limnodynastes peronii, as a model. Specifically, this paper aimed to test: 1) nuclear transplantation with fresh embryonic cells in Limnodynastes peronii and trial the implementation of different egg enucleation methods (pricked eggs only, UV only, and pricked + UV), 2) use of cryopreserved cells in nuclear transplantation and 3) use single nucleotide polymorphisms to determine if the nuclear genomes of nuclear transplant embryos were derived from the injected donor cell nuclei. With both fresh and cryopreserved embryonic cells as donor cell nuclei, development of the nuclear transplant embryos was low compared to the fertilized controls. Only one nuclear transplant embryo derived from a fresh embryonic cell (injected into a UV only enucleated egg) developed into a tadpole. This attained stage 36 of the Gosner Staging System for Anurans (hind limbs) before dying. As an egg activation technique, pricking the eggs resulted in higher cleavage rates and development to blastula than UV only and pricked + UV nuclear transplants (in trials with cryopreserved cells), but none of the nuclear transplants from the pricked only group developed beyond blastula. None of the nuclear transplants derived from cryopreserved embryonic cells developed to tadpoles. The most advanced stage attained from cryopreserved embryonic cells was stage 13 (neurula) from UV only eggs. Single nucleotide polymorphism (SNP) analysis testing for genotype mismatches was used to determine if the genomes of developing nuclear transplant embryos were derived from the injected donor cell nucleus. SNP analysis confirmed that two true clones were generated in this study. This is the first confirmed nuclear transplantation in any Australian native frog.

developmental biology↗

bioRxiv2025

Goldilocks conundrum explains cryoinjury in slow-cooled amphibian embryonic cells

Cryopreservation of intact fish and amphibian eggs and embryos is challenging due to the sheer size and yolk content, which prevents proper dehydration and causes lethal intracellular ice formation during cooling. Alternatively, cryopreservation of dissociated embryonic cells allows to biobank diploid genomes. However, amphibian and fish embryos have cells of varying sizes throughout the developing embryo and cell size distribution remains understudied in cryopreservation. This study examined cell size effects during cryopreservation of dissociated embryonic cells from two amphibian species. Most work used Limnodynatses peronii blastula, gastrula, and neurula cells cryopreserved with 10% dimethyl sulfoxide (DMSO) and sucrose at 0%, 1%, or 10%. Increasing sucrose concentration improved post-thaw recovery of membrane intact cells and cell concentrations, with gastrula and neurula cells having better recovery than blastula cells. An interaction amongst cryoprotectant concentration, cell size, and embryonic stage was observed. Higher sucrose improved recovery of larger cells, but reduced recovery of smaller cells. These results supported a "Goldilocks" model of cryoinjury, in which larger cells require more time to dehydrate adequately, while smaller cells are damaged by excessive dehydration and solute effects, implying that optimal cryoprotectant conditions differ by cell size. Based on post-thaw recovery, 10% DMSO + 10% sucrose was optimal and successfully applied to the cryopreservation of neurula cells from the threatened Rawlinsonia littlejohni, marking the first report of embryonic cell cryopreservation in a threatened amphibian. Our results demonstrated the importance of cell size effects on cryoinjury and their consideration in the application of cryopreservation for amphibian conservation. In brief: Since intact amphibian eggs and embryos cannot be cryopreserved, dissociated embryonic cell cryopreservation provides an alternative avenue for biobanking embryonic diploid genomes; however, cell size effects on cryoinjury is poorly understood and the developing amphibian embryo consists of different sized cells. We demonstrated that cell size influences cryopreservation outcomes by cryopreserving embryonic cells from three developmental stages under varying cryoprotectant concentrations.

developmental biology↗

bioRxiv2025

Peri-operative pregabalin does not adversely impact experimentaloutcomes in rat models of chronic pain

IntroductionMultiple mechanisms contribute to the experience of pain where the use of model organisms to dissect mechanistically sensory regulatory circuitry is a vital component of discovering underlying causes of persistent pain in disease states. Such disease states can be modelled in animals using surgical procedures that, ethically, should involve administration of analgesia. However, since basic pain researchers often wish to measure pain-related events, animals may be denied peri-operative analgesia to avoid adversely influencing experimental outcomes. MethodsWe conducted a systematic review of peri-operative analgesia usage in rat spinal nerve ligation (SNL) and cancer-induced bone pain (CIBP) models. Using a combination of behavioural testing and in vivo electrophysiology in the dorsal horn of the spinal cord, we assessed the impact of peri-operative pregabalin on nociceptive behaviours in the acute recovery phase, and behavioural and electrophysiological experimental outcomes in the chronic phase, of rat SNL and CIBP models. ResultsA literature search revealed that, for studies using rat models of SNL or CIBP, only 5.37 % and 12.69 % respectively reported the use of peri-operative analgesia. We then demonstrated that the use of pregabalin as a peri-operative analgesic reduced mechanical hypersensitivity in the acute period following SNL surgery, with no impact on behavioural, electrophysiological or neuropharmacological outcomes in the chronic phase of either model. ConclusionsThis study challenges the basic science researchers reasoning that peri-operative analgesia confounds neurobiological outcomes. The use of peri-operative analgesia should be an important consideration to improve animal welfare in chronic models of pain. SummaryPeri-operative analgesia can improve animal welfare without adversely affecting long term behavioural or pharmacological experimental outcomes in chronic pain models.

neuroscience↗

bioRxiv2025

Transcriptional Profiling of Commonly Used Liver Cancer Cell Lines Reveals Disease-Specific Modeling Potential and Authentication Concerns

Cell lines are essential tools for liver cancer research, yet their molecular fidelity to primary tumors remains incompletely characterized. Here we comprehensively evaluated transcriptomic similarities between commonly used liver cancer cell lines and primary tumor subtypes to guide optimal model selection. We analyzed RNA sequencing data from 541 samples spanning primary HCC, HPBL, CHOL, and FLC tumors, alongside 21 liver cancer cell lines and primary human hepatocytes. Through systematic variance analysis, we identified 2,523 highly variable genes distinguishing cancer subtypes and cell lines, then performed correlation analyses, unsupervised clustering, and pathway enrichment to assess molecular similarities. Molecular subtypes within each cancer type were identified through hierarchical clustering and characterized using pathway analysis. HepG2 cells showed strongest correlation with HPBL (r=0.62), confirming their hepatoblastoma origin despite frequent HCC misclassification. This correlation was driven by shared Wnt pathway dysregulation signatures. Huh7 cells best represented HCC, particularly the immune-modulatory, MYC-activated subtype with the highest median correlation. RBE cells optimally modeled CHOL, specifically the dedifferentiated, immune-evasive subtype. Several commonly used cell lines (LO2, SMMC-7721, MHCC97L) and specific publicly available samples demonstrated likely HeLa contamination. Primary human hepatocytes cultured under physioxic conditions better preserved liver-specific transcriptional programs compared to standard culture. No established cell line analyzed represented FLC strongly, identifying the need for a standard, available model. This transcriptomic framework provides evidence-based guidance for selecting appropriate liver cancer cell line models and highlights the critical need for rigorous cell line validation to improve experimental design and translational relevance of liver cancer research.

cancer biology↗

bioRxiv2025

Genetic background influences the effects of RNAi-by-feeding for DNA methyltransferases in Daphnia pulex.

DNA methyltransferases (DNMTs) are well-characterized epigenetic enzymes responsible for transferring methyl groups to and from DNA. Three main DNMT orthologues differ in function and methylation capability. They each are evolutionarily conserved across diverse taxa, but few studies investigate them jointly. We did so in Daphnia, freshwater microcrustaceans used extensively in research on genetic diversity, phenotypic plasticity, and maternal effects. Most Daphnia reproduce asexually through cyclic parthenogenesis, making the Daphnia system an ideal choice for studying epigenetic phenomena. Advances in gene expression control techniques, including RNA interference (RNAi), have increased the versatility and power of the Daphnia system. RNAi is a post-transcriptional gene silencing mechanism that operates through sequence-specific cleavage of endogenous messenger RNA (mRNA) transcripts. Here, we used an RNAi bacterial feeding regime to target the three DNA methyltransferase genes in two clones of Daphnia pulex. We observed significant genotypic differences in response to the RNAi feeding regime, namely, the mortality of one clone. In the other, DNMT expression significantly increased in five of the six experimental treatments, with the highest level observed in animals treated with the GFP double-stranded RNA bacterial vector control. However, DNMT expression was reduced in all three DNMT RNAi treatments relative to the GFP control. Furthermore, we found strong cross-reactivity, where targeting one DNMT resulted in the reduction of the other two. This response may be associated with known immune pathways involving signal transduction that can be stimulated by viral and bacterial signals, or it may result from previously unknown aspects of DNMT biology.

genetics↗

bioRxiv2025

Persistent chromatin loops shape gene expression plasticity upon stimulation and restimulation of human neurons

Persistent molecular correlates of long-term memory storage remain an open question. Here, we stimulate and re-stimulate human neurons and use multi-modal single-nucleus technologies to query DNA methylation, higher-order chromatin folding, and gene expression. We find enduring traces of activity-gained and activity-lost chromatin loops. Genes anchoring persistent activity-gained loops exhibit activity-upregulated expression, whereas persistent activity-lost loops anchor activity-downregulated genes that remain repressed five days post-stimulation. CTCF-bound looped enhancers and promoters are refractory to activity-dynamic DNA methylation. Looped enhancers bound by CTCF can exhibit memory of activity-induced histone modifications and persistent expression of activity-upregulated genes. Upon second stimulation, activity-upregulated genes are robustly re-induced when unlooped but remain nonresponsive at persistent loops akin to habituation. Activity-independent gene expression can be downregulated when unlooped but protected from homeostatic downscaling when anchored in persistent loops. Our data reveal long-term genome folding persistence linked to plasticity of activity-dependent gene expression during recall in human neurons. Structured AbstractO_ST_ABSIntroductionC_ST_ABSA long-standing question in neuroscience is how memories of previous experiences are stored in the mammalian brain to facilitate recall over the lifetime of an individual. Classic models of memory posit that both synapse-specific events and cell-wide transcriptional programs are required for encoding, consolidation, and long-term storage of memory (1, 2). Learning involves synapse strengthening, and synapse weakening has been linked to memory loss (3, 4). Nascent transcription and protein synthesis also occur in response to neural activation in vitro and in vivo (5-7). A leading hypothesis, the synaptic tagging and capture model, asserts that specific synapses are biochemically marked during encoding and newly made cell-wide RNA/proteins act specifically on tagged synapses to maintain long-term memory (8). Multiple historic studies have pursued the identification of proteins linked to potentiated synapses (9-13), but few explore the possibility of persistent, activity-dependent patterns of DNA, chromatin, higher-order chromatin folding, or RNA in human models of long-term memory. Over the last decade, the molecular technique of Chromatin-Conformation-Capture has been employed to discover that the mammalian genome folds into thousands loops of (5, 14-28). Loops bring distal non-coding cis regulatory elements into contact with their target genes to influence gene expression (29). They form via the processive motion of the cohesin ring along chromatin until it stalls at the architectural protein CTCF, thus extruding out the intervening DNA (22, 30-32). Loops can markedly reconfigure during lineage commitment in development and in response to genetic perturbations, and their formation is critical for spatiotemporal regulation of expression (22, 30, 33-41). RationaleMultiple recent works suggest that loops connecting activity-dependent enhancers to distal genes and can be induced during neural stimulation in vitro and behavior paradigms in vivo (5, 27, 42, 43). Genetic elimination of the architectural proteins CTCF and cohesin prior to learning substantially impairs memory encoding across multiple behavior tasks in vivo (42, 44, 45). Cohesin-mediated loops are necessary for the establishment of new gene expression programs in post-mitotic neurons, including the upregulation of genes encoding axon guidance, dendritic spine morphology, and synaptic plasticity during neuron maturation in vivo and activity-dependent gene expression during neural stimulation in vitro (27). Fear conditioning recruits epigenetically plastic neurons to form the memory engram (46) and induces chromatin accessibility changes at enhancers that might persist at least five days after fear conditioning (43). Together, these data provide the rationale for our hypothesis that key structural features of higher-order chromatin folding could be persistent long after the exposure and removal of a pharmacological, environmental, or behavioral stimulus that causes neurons to fire action potentials. ResultsHere, we apply cutting-edge single-nucleus multi-modal technologies toward the goal of ascertaining the extent to which there are enduring traces of higher-order chromatin folding, DNA methylation, histone modifications at activity-dependent enhancers, and/or mRNA levels in single human neurons upon exposure to a repetitive stimulation and re-stimulation paradigm. We find that pharmacological stimulation of human induced pluripotent stem cell (iPSC)-derived neurons induces chromatin loop plasticity, including persistent activity-gained (PG) and persistent activity-lost (PL) loops that are dependent on CREB phosphorylation. We also query activity-dependent patterns of DNA methylation genome-wide and demonstrate that the majority of looped enhancers and promoters are negligibly differentially methylated in human neurons in response to pharmacological stimulation. CTCF binding to looped enhancers and promoters further protects such regulatory elements from activity-stimulated changes in DNA methylation. What are the potential functional effects of persistent chromatin loops? We find that activity-lost persistent loops anchor activity-downregulated genes that remain repressed five days post-stimulation. By contrast, activity-gained persistent loops are enriched for activity-induced gene expression at 2-5 hours post-stimulation, but mRNA levels resume baseline levels by five days after removal of the stimulus. A subset of promoter-enhancer loops bound by CTCF can exhibit enduring traces of the histone modification H3K27ac. Promoters persistently looped to CTCF-bound persistent activity-induced enhancers can exhibit memory of activity-upregulated gene expression for at least five days after stimulation. Finally, we re-stimulated our human neurons five days after the original stimulation event and assessed gene expression with single-nucleus RNA-seq. Upon second stimulation, we unexpectedly find that activity-upregulated genes can be robustly re-induced when unlooped but remain nonresponsive at persistent loops akin to habituation. We also unexpectedly find that activity-independent, invariant gene expression can be downregulated at second stimulation when unlooped but protected from homeostatic downscaling when anchored in persistent loops. ConclusionTaken together, our data reveal enduring traces of higher-order chromatin loops in human neurons after activity-simulation and their link to gene expression plasticity and habituation during re-stimulation and re-stimulation of human neurons. Our work sheds light on the role for chromatin and gene expression in an important unsolved paradox in neuroscience: How is information encoded in neural circuits on the timescale of years despite the rapid turnover of synaptic proteins/RNAs in hours to weeks? Persistent chromatin loops shape plasticity in cell-wide RNA levels, thus laying the foundation for future studies linking genes identified here to their subcellular localization and possible functional role at the synapse.

neuroscience↗

bioRxiv2025

Site-Specific Nanobody Inhibitors of the Proteasomal Deubiquitinase UCH37

Dysregulation of the ubiquitin (Ub) proteasome system (UPS) is linked to numerous human diseases, making its components attractive therapeutic targets. Among these, the proteasomal deubiquitinase UCH37 (also known as UCHL5) has two distinct Ub-processing activities: C-terminal Ub hydrolysis and K48-linkage-specific chain debranching. Separating these activities has not been possible with existing small-molecule inhibitors, which target the catalytic site and broadly affect both functions along with many other DUBs. Here, we report the development of two site-specific nanobody inhibitors of UCH37 that together enable this separation. Using yeast surface display, we generated Nb-cS1, which binds the canonical Ub-binding (S1) site and blocks both activities, and Nb-ncS1, which binds the non-canonical K48-specific (ncS1) site and selectively impairs chain debranching without affecting C-terminal Ub hydrolysis. X-ray crystallography and hydrogen-deuterium exchange mass spectrometry define the distinct binding surfaces engaged by each nanobody. In cells, both nanobodies selectively engage UCH37, with Nb-ncS1 showing the cleanest target engagement. These tools provide the first means to independently interrogate UCH37s two enzymatic activities and offer new approaches for dissecting the distinct roles of UCH37 in proteasomal and chromatin-associated contexts.

biochemistry↗

bioRxiv2025

CD33 and Clusterin Interact Biophysically and Genetically to Modulate Alzheimer Risk

We report the results of structural, functional and genetic studies on the CD33 sialic acid- binding receptor that reveal how non-coding variants in CD33 alter risk for Alzheimers disease (AD). The full-length CD33M isoform, whose expression is upregulated by non-coding AD-risk alleles, preferentially forms dimers at the cell surface, where they interact with AD-related proteins (clusterin and A{beta}). This interaction induces CD33M inhibitory signalling and downregulates protective microglial functions including phagocytic removal of amyloid plaques. Human brain expression quantitative trait loci (eQTL) and causal mediation analyses confirm that quantitative interactions between CLU and CD33 genotypes modulate AD phenotypes and suggest that genotypes at these loci might be used to personalise future therapeutic approaches. Our work also highlights several other unexpected aspects of CD33 biology, including a soluble shed extracellular fragment of CD33M and a similar soluble secreted product arising from a truncating mutation in the CD33 extracellular domain (CD33M{Delta}4bp).

neuroscience↗

bioRxiv2025

Dual Bmp-negative feedback loops modulate both AER and ZPA function to buffer and constrain postaxial digit number.

Several lines of evidence indicate that posterior (postaxial) digit number in tetrapod vertebrates is constrained to the pentadactyl state by interactions between the 2 major signaling centers that organize digit pattern and growth, the Shh-expressing ZPA and the Fgf-expressing AER ectoderm. Negative short-range effects of Shh on the immediately overlying AER limit its posterior extent and function, either by direct Shh signaling or with Bmps playing a key role. How this strong inhibitory effect is counter-balanced to maintain pentadactyly in many vertebrate species remains less clear. In this study we used genetic approaches in mouse to re-evaluate the mechanism by which Shh signaling modulates AER function and demonstrate that this occurs via Shh-target Bmps that act as a relay signal, rather than by direct Shh action. Furthermore, we show that Shh-induced Bmps also act directly on the ZPA, in a negative feedback loop, to down-regulate Shh expression and ZPA extent. We provide evidence that these dual Bmp-driven negative feedback loops robustly balance total Bmp levels to constrain postaxial digit number. Significance StatementThis study examines how vertebrates, including humans, are generally constrained to forming five fingers or toes (pentadactyly) during normal development. Two key signaling centers in the limb bud, producing Sonic hedgehog (Shh) in the posterior mesoderm (ZPA) and Fgfs in the marginal ectoderm (AER), interact in a positive feedback loop to direct and coordinate limb outgrowth. However, short-range negative interaction between these two centers limits posterior digit numbers. Using genetic approaches in mouse, we show that Bmps, rather than Shh, signal directly to the AER to act as the primary mediators of this constraint. Shh-induced target Bmps act directly on AER to inhibit its function and prevent posterior digit expansion, and they also act directly on ZPA in a negative feedback loop to inhibit Shh expression. These dual Bmp-driven feedback circuits act together to balance Bmp activity and robustly limit posterior digit number to the pentadactyl state. This finding enhances our understanding of how disrupted developmental regulation may lead to congenital limb malformations and how evolutionary constraints on digit number may be imposed.

developmental biology↗

bioRxiv2025

IsoDGR-Induced Endothelial Cytoskeletal Disruption Drives Age-Related Blood-Brain Barrier Breakdown

Brain aging is characterized by progressive breakdown of the blood-brain barrier (BBB), which correlates with neuroinflammation and cognitive decline. Emerging evidence implicates degenerative modifications of the vascular proteins as a key driver of BBB dysfunction. In particular, spontaneous deamidation of Asp-Gly-Arg (NGR) motifs generates isoAsp-Gly-Arg (isoDGR) sequences that structurally mimic canonical Arg-Gly-Asp (RGD) integrin-binding ligands. Here, we show that age-associated accumulation of isoDGR in the brain cortex induces endothelial cytoskeletal collapse and tight junction disorganization, leading to BBB breakdown. Using mice lacking the L-isoaspartyl repair enzyme PCMT1 (which accelerates isoDGR accumulation) and wild type aged mice, we found markedly elevated isoDGR in brain tissues accompanied by focal microhemorrhages and increased BBB permeability. Recent whole-genome sequencing suggests that a common PCMT1 variant is linked to neurodegenerative disease risk, indicating potential clinical relevance in vascular aging. Remarkably, systemic treatment with an isoDGR-neutralizing antibody largely prevented capillary breaches and leakage, and even restored barrier integrity in aged wild-type mice. To uncover the molecular mechanism, we exposed brain endothelial cells to synthetic isoDGR-peptides, which recapitulated these effects. Unbiased RNA-sequencing reinforced these findings, revealing broad transcriptomic reprogramming of cytoskeletal, cell-cell junction, inflammatory, and stress-response pathways. Functional studies demonstrated that isoDGR triggered collapse of F-actin stress fibers, disrupted junctional ZO-1 and VE-cadherin, increased monolayer permeability to macromolecules, and impaired endothelial cell migration and proliferation. IsoDGR-treated endothelial cells exhibited increased oxidative stress, upregulation of ICAM-1/VCAM-1/CCL-2, and adopted a senescent phenotype. Our results suggest that isoDGR hijacks endothelial integrin signaling to destabilize the actin cytoskeleton and tight junctions, a process that breaches the BBB and subsequently activates inflammatory and senescence programs. In summary, we identify BBB disruption via isoDGR-induced cytoskeletal dysfunction as a central pathology of vascular aging, and demonstrate that targeting isoDGR damage preserves BBB integrity and attenuates neuroinflammation.

neuroscience↗

bioRxiv2025

Genes on the Move: Exploring the Resistome of Viable Isolates from a First Nation Community in Manitoba

Despite wastewater treatment plants (WWTPs) and oxidation lagoons (OL) being designed for pollutant and pathogen removal, these systems can also serve as environments conducive to the exchange of mobile genetic elements (MGEs) and antibiotic resistance genes (ARGs) among microorganisms. This context facilitates the dissemination of antimicrobial resistance (AMR) among both pathogenic and non-pathogenic environmental bacteria, as well as phage populations, within these facilities. To enhance understanding of AMR dynamics in wastewater, our study examined the resistome of bacterial isolates recovered from a OL in a First Nation (FN) community in Manitoba, Canada. Thirty-five samples were collected from all OL stages, including raw sewage, lagoons, submerged attached growth reactor (SAGR), UV-treated effluent, and an upstream control site, from September 2022 to April 2023. Whole genome sequencing of 58 viable isolates was performed using Oxford Nanopore MinION, with genomes reconstructed via de novo assembly. The isolates corresponded to the following genera: Aeromonas 50.87% , Serratia 15.78%, Pantoea and Escherichia 7.01% each, Lelliottia 5.26%, Rahnella, Enterobacter and Buttiauxella 3.50% each, and finally Acinetobacter, Yersinia and Citrobacter 1.75%. Furthermore, a total of 32,559 elements were classified as ARGs. MGEs carrying ARGs were detected in 72.72% of the isolates. ARGs conferring resistance to macrolides, tetracyclines, and fluoroquinolones demonstrated a notable presence within MGEs (plasmids and bacteriophages) identified throughout the facilitys stages. This research represents a non-invasive approach, enabling an in-depth exploration of AMR dynamics by discerning the resistome profiles of bacteria residing within a rural OL facility from a FN community. ImportanceOLs commonly found in rural and Indigenous communities in Canada, are crucial for mitigating environmental hazards and safeguarding public health. Paradoxically, these systems can also serve as hotspots for the dissemination of ARGs and MGEs, thereby exacerbating AMR. The persistence of ARGs within both pathogenic and non-pathogenic bacteria, often carried on MGEs including phages, in treated effluents raises significant concerns about the environmental impact and public health. This study offers fundamental insights into the unique dynamics of AMR within OLs serving a First Nation community in Manitoba. Understanding these specific dynamics is crucial for developing robust monitoring frameworks and implementing targeted, sustainable strategies to mitigate the public health and environmental risks posed by AMR in these often-underserved remote settings.

microbiology↗

bioRxiv2025

Adrenergic Hypersensitivity Drives Ventricular Arrhythmias Following Loss of Plexin-Mediated Cardiac Innervation

BackgroundVentricular arrhythmias (VAs) are a leading cause of death and arise from a combination of cardiac muscle injury and dysfunction of the intramyocardial sympathetic nerves that control cardiac electrophysiology. The adrenergic mechanisms by which intramyocardial nerves contribute to arrhythmogenesis are poorly understood. Semaphorin-plexin signaling pathways are responsible for developmental guidance of sympathetic nerves onto the heart and have previously been associated with VAs in humans. ObjectiveTo investigate adrenergic control of arrhythmogenesis, we probed the cardiac electrophysiology of a Plexin-A3/-A4 double knockout mouse with loss of cardiac adrenergic nerves. MethodsWe studied cardiac structure and function using tissue clearing, immunohistochemistry, and echocardiography. We utilized ECG and optical mapping of action potentials to evaluate electrophysiologic responses to pharmacologic beta ({beta})-adrenergic stimulation and blockade. We measured circulating catecholamines and quantified {beta}-adrenergic receptor ({beta}AR) density in cardiac membranes. Finally, we performed a phenome-wide association study utilizing data from the UK Biobank to search for associations between PLXNA4 and human arrhythmias. ResultsMice with loss of plexin-dependent cardiac innervation had structurally normal hearts but displayed spontaneous VAs driven by adrenergic hypersensitivity, as well as increased cardiac {beta}AR density. Several human PLXNA4 variants were associated with arrhythmia phenotypes. ConclusionThese data establish a model of VAs driven by enhanced adrenergic receptor signaling, in the absence of structural heart disease, which can be used to investigate adrenergic mechanisms of arrhythmogenesis and to identify novel antiarrhythmic targets.

physiology↗

bioRxiv2025

Determining preclinical safety of Aclarubicin in pediatric malignancies

BackgroundAnthracyclines are among the most effective chemotherapeutic agents used to treat pediatric malignancies. However, their clinical use is limited by dose-dependent toxicities, particularly cardiotoxicity and secondary malignancies. Aclarubicin (Acla) is an anthracycline derivative that induces chromatin damage while sparing DNA, offering potential therapeutic benefit with reduced longterm toxicity. MethodsWe evaluated the anti-tumor efficacy and safety profile of Acla in multiple in vitro pediatric cancer models and in vivo mouse models designed to mimic anthracycline re-treatment following prior doxorubicin (Doxo) exposure. Tumor growth, genotoxic stress, survival, and organ toxicity were assessed. ResultsAcla demonstrated robust anti-tumor activity comparable to Doxo across diverse pediatric in vitro models. Unlike Doxo, Acla treatment did not induce significant genotoxic stress. In vivo, mice receiving Acla after Doxo exposure showed no evidence of cumulative cardiotoxicity or end-organ damage. In contrast, a second course of Doxo led to significant toxic mortality, but was surprisingly not attributable to classic cardiac injury. ConclusionOur study highlights Acla as a promising anthracycline derivative for pediatric cancers, with potent anti-tumor efficacy and a superior safety profile, even following prior anthracycline exposure. These results support continued investigation of chromatin-damaging anthracyclines that can kill pediatric cancer cells without inducing genotoxic stress. In addition, our studies underscore the need to refine preclinical models to better understand both acute and chronic anthracycline toxicities in pediatric and adolescent populations.

cancer biology↗

bioRxiv2025

Gut sulfide metabolism modulates behavior and brain bioenergetics

The host-microbiome interface is rich in metabolite exchanges and exquisitely sensitive to diet. Hydrogen sulfide (H2S) is present at high concentrations at this interface, and is a product of both microbial and host metabolism. The mitochondrial enzyme, sulfide quinone oxidoreductase (SQOR), couples H2S detoxification to oxidative phosphorylation; its inherited deficiency presents as Leigh disease. Since an estimated two thirds of systemic H2S metabolism originates in gut, it raises questions as to whether impaired sulfide clearance in this compartment contributes to disease, and whether it can be modulated by dietary sulfur content. In this study, we report that SQOR deficiency confined to murine intestinal epithelial cells, perturbs colon bioenergetics that is reversed by antibiotics, establishing a significant local contribution of microbial H2S to host physiology. We also find that a 2.5-fold higher methionine intake, mimicking the difference between animal and plant proteins, synergized with intestinal SQOR deficiency to adversely impact colon architecture and alter microbiome composition. In serum, increased thiosulfate, a biomarker of H2S oxidation, revealed that intestinal SQOR deficiency combined with high dietary methionine, affects sulfide metabolism globally and perturbs energy metabolism as indicated by higher ketone bodies. The mice exhibited lower exploratory locomotor activity while brain MRI revealed an atypical reduction in ventricular volume, which was associated with lower aquaporin 1 that is important for cerebrospinal fluid secretion. Our study reveals the dynamic interaction between dietary sulfur intake and sulfide metabolism at the host-microbe interface, impacting gut health, and the potential for lower dietary methionine intake to modulate pathology. Significance StatementThe host-microbiome interface is rich in metabolite-based communications that are modulated by diet. Hydrogen sulfide (H2S), which is a respiratory poison at high concentrations, is enriched at this interface, and is detoxified by the host enzyme, sulfide quinone oxidoreductase (SQOR). Given the quantitatively significant contribution of gut to systemic H2S metabolism, we examined how SQOR deficiency restricted to murine intestinal epithelial cells, interacts with high dietary methionine, designed to approximate the difference between plant versus animal protein levels, to affect local and global bioenergetics. Our study revealed profound short- and long-range impacts resulting from the synergy between decreased H2S clearance capacity in gut and high dietary methionine on global energy metabolism, brain pathology, and behavior.

biochemistry↗