Biology subjects

Singh, V.

Publications and source records attributed to Singh, V..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Pathological Mechanisms of Motor Dysfunction in Familial Danish Dementia: Insights from a Knock-In Rat Model

Familial Danish Dementia (FDD) is a rare autosomal dominant neurodegenerative disorder caused by a mutation in the integral membrane protein 2B (ITM2b) gene. Clinically, FDD is characterized by cerebral amyloid angiopathy (CAA), cerebellar ataxia, and dementia. Notably, FDD shares several neuropathological features with Alzheimers disease (AD), including CAA, neuroinflammation, and neurofibrillary tangles. In this study, we investigate the pathological mechanisms linking CAA, white matter damage, and motor dysfunction using a recently developed FDD knock-in (FDD-KI) rat model. This model harbors the Danish mutation in the endogenous rat Itm2b gene, along with an App gene encoding humanized amyloid-{beta} (A{beta}). Our analysis revealed substantial vascular Danish amyloid (ADan) deposition in the cerebellar subpial and leptomeningeal vessels of FDD-KI rats, showing an age-related increase comparable to that observed in human FDD patients. Additionally, vascular A{beta} deposits (A{beta}-CAA) were present in FDD-KI rats, but A{beta}-CAA patterns showed some differences between species. Motor function assessments in FDD-KI rats demonstrated age-accelerated motor deficits and gait abnormalities, mirroring the clinical characteristics of FDD patients. To further explore the mechanisms underlying these deficits, we examined cerebellar pathology and found age-related myelin disruption and axonal fiber loss, consistent with postmortem human FDD pathology. Cerebellar demyelination appeared to be driven by neuroinflammation, marked by increased microglial/macrophage activation in response to vascular amyloid deposition. Additionally, we observed extravascular fibrinogen leakage, indicating widespread vascular permeability in both white and gray matter, with fibrinogen deposits surrounding amyloid-positive vessels in aged FDD-KI rats and postmortem FDD cerebellum. These findings suggest that this FDD-KI rat model is the first animal model to recapitulate key neuropathological features of human FDD patients, including both ADan- and A{beta}-type CAA, neuroinflammation, and white matter lesions--pathologies that may underlie the motor and gait impairments seen in the disease.

neuroscience↗

bioRxiv2025

Revealing the impact of Pseudomonas aeruginosa quorum sensing molecule 2'-aminoacetophenone on human bronchial-airway epithelium and pulmonary endothelium using a human airway-on-a-chip

Pseudomonas aeruginosa (PA) causes severe respiratory infections utilizing multiple virulence functions. Our previous findings on PA quorum sensing (QS)-regulated small molecule, 2-aminoacetophenone (2-AA), secreted by the bacteria in infected tissues, revealed its effect on immune and metabolic functions favouring a long-term presence of PA in the host. However, studies on 2-AAs specific effects on bronchial-airway epithelium and pulmonary endothelium remain elusive. To evaluate 2AAs spatiotemporal changes in the human airway, considering endothelial cells as the first point of contact when the route of lung infection is hematogenic, we utilized the microfluidic airway-on-chip lined by polarized human bronchial-airway epithelium and pulmonary endothelium. Using this platform, we performed RNA-sequencing to analyse responses of 2-AA-treated primary human pulmonary microvascular endothelium (HPMEC) and adjacent primary normal human bronchial epithelial (NHBE) cells from healthy female donors and potential cross-talk between these cells. Analyses unveiled specific signaling and biosynthesis pathways to be differentially regulated by 2-AA in epithelial cells, including HIF-1 and pyrimidine signaling, glycosaminoglycan, and glycosphingolipid biosynthesis, while in endothelial cells were fatty acid metabolism, phosphatidylinositol and estrogen receptor signaling, and proinflammatory signaling pathways. Significant overlap in both cell types in response to 2-AA was found in genes implicated in immune response and cellular functions. In contrast, we found that genes related to barrier permeability, cholesterol metabolism, and oxidative phosphorylation were differentially regulated upon exposure to 2-AA in the cell types studied. Murine in-vivo and additional in vitro cell culture studies confirmed cholesterol accumulation in epithelial cells. Results also revealed specific biomarkers associated with cystic fibrosis and idiopathic pulmonary fibrosis to be modulated by 2-AA in both cell types, with the cystic fibrosis transmembrane regulator expression to be affected only in endothelial cells. The 2-AA-mediated effects on healthy epithelial and endothelial primary cells within a microphysiological dynamic environment mimicking the human lung airway enhance our understanding of this QS signaling molecule. This study provides novel insights into their functions and potential interactions, paving the way for innovative, cell-specific therapeutic strategies to combat PA lung infections.

molecular biology↗

bioRxiv2025

Future flooding tolerant rice germplasm: resilience afforded beyond Sub1A gene

Developing high-yielding, flood-tolerant rice varieties is essential for enhancing productivity and livelihoods in flood-prone ecologies. We explored genetic avenues beyond the well-known SUB1A gene to improve flood resilience in rice. We screened a collection of 6,274 elite genotypes from IRRIs germplasm repository for submergence and stagnant flooding tolerance over multiple seasons and years. This rigorous screening identified 89 outstanding elite genotypes, among which thirty-seven exhibited high submergence tolerance, surpassing the survival rate of SUB1A introgression genotypes by 40-50%. Thirty-five genotypes showed significant tolerance to stagnant flooding, and 17 demonstrated dual tolerance capabilities, highlighting their adaptability to varying flood conditions. The genotypes identified have a broader genetic diversity and harbor 86 key QTLs and genes related to traits such as grain quality, grain yield, herbicide resistance, and various biotic and abiotic traits, highlighting the richness of the identified elite collection. Besides germplasm, we introduce an innovative breeding approach called Transition from Trait to Environment (TTE). TTE leverages a parental pool of high-performing genotypes with complete submergence tolerance to drive population improvement and enable genomic selection in the flood breeding program. Our approach of TTE achieved a remarkable 65% increase in genetic gain for submergence tolerance, with the resulting fixed breeding genotypes demonstrating exceptional performance in flood-prone environments of India and Bangladesh. The elite genotypes identified herein represent invaluable genetic resources for the global rice research community. By adopting the TTE approach, which is trait agonistic, we establish a robust framework for developing more resilient genotypes using advanced breeding tools. Plain Language SummaryTo address climate challenges, an urgent focus is necessary to identify and develop flood-tolerant rice varieties, particularly for flood-prone ecosystems across Asia and Africa. We screened 6,274 elite genotypes from IRRIs germplasm and identified 89 promising lines with improved tolerance to submergence and stagnant flooding. Among these, 37 demonstrated 40-50% greater submergence tolerance than SUB1A introgression lines, 35 exhibited stagnant flooding tolerance, and 17 showed dual tolerance. These genotypes contain 86 key QTLs and genes associated with yield, grain quality, and biotic and abiotic tolerance traits. A new breeding strategy, the Transition from Trait to Environment (TTE) approach, was developed. We achieved a genetic gain of 65% for submergence tolerance in rice using this method. The newly identified germplasm provides invaluable genetic resources for the global rice research community to develop flood-tolerant rice genotypes. Core ideas The SUB1A gene, enabling rice to survive underwater for 14 days, marked a significant breakthrough. We have identified elite genotypes with submergence tolerance significantly surpassing the SUB1A gene-mediated tolerance. The diverse elite genotypes identified harbor 86 key genes and QTLs that affect various traits positively. Developed a unique breeding strategy for implementing population improvement in challenging environments. The new breeding strategy demonstrated a genetic gain of 65% for submergence tolerance.

plant biology↗

bioRxiv2025

Receptor tyrosine kinase AXL regulates Golgi organization and function through an adhesion-Arf1 signaling axis in breast and lung cancers

Cell-matrix adhesion regulates membrane trafficking, Golgi organisation and function. Differential Golgi organisation in cancer cells could drive changes in trafficking and processing of cargoes. A simple screen evaluating Golgi organisation identifies breast (MDAMB231 vs MCF7) and lung cancer (A549 vs CaLu1) cell line pairs with differently organised Golgi, regulated differentially by loss of adhesion. In silico analysis of differentially expressed genes in the CCLE database, evaluated for their association with the Golgi in interaction networks and literature, identified AXL as a putative regulator of Golgi in these cancers. AXL prominently localized at the Golgi, undergoes displacement from the Golgi when inhibited by R428 and knocked down using siRNA, causing the Golgi to disorganize. AXL-dependent regulation of Golgi organisation is also dependent on cell-matrix adhesion. AXL binds active Arf1, whose recruitment to the Golgi is vital for its organisation. On loss of adhesion, loss of AXL and active Arf1 from the Golgi, drives its disorganization to affect Golgi-dependent microtubule acetylation and cell surface glycosylation in MDAMB231 and A549 cells respectively. Together, this validates our screen to identify novel regulators of the Golgi, and identifies AXL-Arf1 crosstalk as a vital mediator of its organisation and function in cancer cells.

cell biology↗

bioRxiv2025

Partially differentiated enterocytes in ileal and distal-colonic human F508del-CF-enteroids 1 secrete fluid in response to forskolin and linaclotide.

Constipation causes significant morbidity in Cystic Fibrosis (CF) patients. Using CF patient (F508del) derived ex vivo ileal and distal colonic/rectal enteroids as a model and the Forskolin Induced Swelling Assay (FIS), we compared CFTR mediated fluid secretion in human enterocytes across the crypt-villus axis. CFTR expression and FIS decreased as enterocytes differentiated from crypt to become partially differentiated and then mature villus cells. While there was no FIS response in undifferentiated (crypt enterocytes) F508del-CF enteroids, partially differentiated F508del-CF enteroids had a swelling response to forskolin (cAMP) and linaclotide (cGMP) which was [~]48%, and [~]67% of the response in healthy enteroids, respectively and was prevented by a CFTR inhibitor. Also, linaclotide and a general PDE inhibitor independently enhanced combined CFTR-modulator-induced FIS response from partially differentiated F508del-CF enteroids. These findings demonstrate that partially differentiated ileal and distal colonic F508del-CFTR enteroids can be stimulated to secrete fluid by cAMP and cGMP.

physiology↗

bioRxiv2024

Knock-in Kcnh2 Rabbit Model of Long QT Syndrome Type-2, Epilepsy, and Sudden Death

BackgroundLong QT Syndrome Type-2 (LQT2) is due to loss-of-function KCNH2 variants. KCNH2 encodes Kv11.1 that forms a delayed-rectifier potassium channel in the brain and heart. LQT2 is associated with arrhythmias, seizures, sudden cardiac death, and sudden unexpected death in epilepsy (SUDEP). The goal of the study is to develop a translational model that reproduces the neuro-cardiac electrical abnormalities and sudden death seen in people with LQT2. MethodsWe generated the first knock-in rabbit model of LQT2 (Kcnh2(+/7bp-del)), due to a 7 base-pair (7bp) deletion in the pore domain of the endogenous rabbit Kcnh2 gene. ResultsMutant Kcnh2 is expressed in the heart and brain and constitutes 11% of total Kcnh2 in Kcnh2(+/7bp-del) rabbits. Total Kcnh2, WT Kcnh2, and WT Kv11.1 expression is lower in Kcnh2(+/7bp-del) vs. WT rabbits. Kcnh2(+/7bp-del) rabbits exhibit prolonged cardiac ventricular repolarization (QTc, JTec, JTpc). There is an increased prevalence of spontaneous epileptiform activity and clinical seizures in Kcnh2(+/7bp-del) (7 of 37 rabbits) vs. WT rabbits (1:68 rabbits, p<0.003). 18.9% of Kcnh2(+/7bp-del) vs. 1.5% of WT rabbits died suddenly and spontaneously (p<0.003). We recorded 2 spontaneous lethal events in Kcnh2(+/7bp-del) rabbits: (1) sudden cardiac death and (2) seizure-mediated sudden death due to generalized tonic-clonic seizures, post-ictal generalized EEG suppression, bradycardia, ECG-T-wave inversion, focal cardiac activity, and asystole/death. ConclusionsWe developed the first genetic rabbit model of LQT2 that reproduces the cardiac and epileptic phenotypes seen in people with LQT2. Kcnh2(+/7bp-del) rabbits provide a valuable tool for future mechanistic studies, development of neurotherapeutics, and cardiac-safety testing.

physiology↗

bioRxiv2024

Cryo-EM reveals that cardiac IGLV6-derived AL fibrils can be polymorphic

Immunoglobulin light chain (AL) amyloidosis is a systemic disorder caused by the misfolding and aggregation of free immunoglobulin light chains (LCs) secreted by abnormal plasma cells. The resulting amyloid fibrils deposit in multiple organs, leading to progressive dysfunction and increased morbidity and mortality. Despite recent advances, the molecular determinants of LC aggregation and phenotypic variability remain poorly understood. Structural characterization of ex-vivo fibrils provides key insights into these pathogenic processes. Here, we report cryo-electron microscopy structures of cardiac AL amyloid fibrils derived from an IGLV6 light chain. The fibrils display two distinct morphologies composed of single and double protofilaments, each adopting a previously unobserved fold. Comparison with previously reported AL fibril structures reveals that while individual mutations can alter the local conformation, IGLV6-derived fibrils share conserved structural motifs that may underlie common aggregation pathways. These findings expand the disease structural landscape and highlight sequence-dependent yet structurally constrained mechanisms of LC fibril formation.

molecular biology↗

bioRxiv2024

The mitochondrial methylation potential gates mitoribosome assembly

S-adenosylmethionine (SAM) is crucial for cellular processes, primarily serving as the principal methyl group donor of the cell and playing a key role in gene regulation and translation on the ribosome. Inside mitochondria, SAM-dependent methylations occur at several steps of gene expression, but their role and significance remain unclear. Using direct long-read RNA sequencing on mouse tissue and mouse embryonic fibroblasts, we demonstrate that the mitochondrial ribosomal gene cluster is not efficiently processed without mitochondrial SAM. This results in the accumulation of unprocessed ribosomal RNA precursors. Protein profiling of ribosome fractions revealed that these precursors are associated with processing and ribosome assembly factors, indicating stalling at an early stage. Structural analysis of the mitochondrial ribosome by cryogenic electron microscopy revealed that mitochondrial SAM is required during peptidyl transferase centre formation and mitochondrial ribosome assembly. Our data thus identify a critical role for methylation at two steps during mitochondrial gene expression.

molecular biology↗

bioRxiv2024

Caveolin-1 and Aquaporin-4 as Mediators of Fibrinogen-Driven Cerebrovascular Pathology in Hereditary Cerebral Amyloid Angiopathy

Cerebral Amyloid Angiopathy (CAA), characterized by amyloid-{beta} (A{beta}) accumulation within perivascular spaces (PVS), contributes to vascular damage and inflammation in Alzheimers disease (AD). Despite its significance, the mechanisms driving A{beta} deposition in PVS and the resulting vascular pathology remain poorly understood. Growing evidence suggests that fibrinogen, the main component in blood clots, interacts with A{beta} and exacerbates inflammation in AD. Fibrinogen also co-deposits with A{beta} in the PVS of CAA-positive vessels in the brains of hereditary CAA patients. However, the mechanisms by which fibrinogen contributes to cerebrovascular impairment remain poorly understood. To investigate this, we used TgSwDI transgenic mice, which develop robust CAA pathology, and observed a significant increase in fibrin(ogen) extravasation and colocalization with A{beta} in the PVS. Moreover, we observed a significant aquaporin-4 (AQP4) depolarization in CAA-laden blood vessels of TgSwDI mice, which correlated with fibrin(ogen)-A{beta} colocalization. Given AQP4 crucial role in A{beta} clearance through glymphatic pathway, its depolarization may disrupt critical A{beta} clearance, thereby exacerbating CAA pathology. Additionally, Caveolin-1, a protein involved in non-specific transcytosis across the endothelium, significantly increased with age in TgSwDI mice and correlated with fibrin(ogen) extravasation. To further explore the relationship between fibrin(ogen) and these cerebrovascular alterations, we depleted fibrinogen in TgSwDI mice using siRNA approach. This intervention resulted in decreased CAA, restored polarized expression of AQP4, reduced caveolin-1 levels, attenuated microglial activation, and improved spatial memory in fibrinogen-depleted TgSwDI mice. These findings suggest that targeting fibrinogen could be a promising strategy for mitigating CAA pathology and its associated cerebrovascular pathology. Significance StatementOur study uncovers the mechanism by which fibrin(ogen)-A{beta} colocalization exacerbates CAA pathology. Our findings highlight the potential link between fibrinogen/ fibrin(ogen)-A{beta} colocalization and AQP4 depolarization thereby exacerbating CAA pathology. The age-dependent increase of endothelial caveolin-1 could facilitate fibrin(ogen) extravasation, assisting the later to binds to A{beta} in the perivascular space which ultimately induce microglial neuroinflammation and AQP4 depolarization, thus exacerbating CAA pathology. Furthermore, fibrinogen depletion could mitigate CAA severity, reduce microglial activation, restore AQP4 polarization and memory impairment. These results suggest that targeting fibrinogen and caveolin-1-mediated transcytosis may offer new strategies to address CAA-associated cerebrovascular pathology.

neuroscience↗

bioRxiv2024

Identification and characterization of UBA5 inhibitors

UBA5 is a critical E1-activating enzyme in the UFMylation pathway, a post-translational modification process implicated in neurodegenerative diseases and cancers. In this study, we developed a high-throughput screening (HTS) assay to identify inhibitors of UBA5 from a library of blood-brain barrier (BBB)-permeable compounds. The assay, based on the AMP-Glo kit, enabled the identification of five novel UBA5 inhibitors belonging to three distinct chemical scaffolds with low micromolar IC50 values. These inhibitors demonstrated selectivity for UBA5 over other E1 enzymes, such as UBA1, and showed efficacy in inhibiting endogenous UFMylation in HEK293T cells. The identified inhibitors not only provided valuable tools for studying UFMylation but also represent potential therapeutic candidates for diseases associated with dysregulated UFMylation, such as Alzheimers disease and cancer. Further optimization of these compounds is necessary to enhance their potency and pharmacokinetic properties. Our findings highlight UBA5 as a promising therapeutic target and pave the way for the development of selective inhibitors that can modulate UFMylation in human diseases.

biochemistry↗

bioRxiv2024

Mechanistic insights into the inhibition of dengue virus NS5 methyltransferase by herbacetin

Herbacetin (HC) is a naturally occurring flavonoid compound with a dual antiviral mechanism. It inhibits the polyamine biosynthetic pathway and targets the methyltransferase (MTase) enzyme of the dengue and chikungunya virus. However, understanding the detailed inhibition mechanism of DENV MTase by HC remains unclear. The crystal structure of DENV3 nonstructural protein 5 (NS5) MTase in complex with HC and S-adenosyl-L-homocysteine presented in this study gives structural insights into the inhibition mechanism. Structural analysis revealed that HC binds to the cap 0 RNA site near the GTP binding site in the DENV3 NS5 MTase, and fluorescence polarization assay demonstrated HC inhibits GTP binding with an inhibition constant Ki value of [~] 0.43 {micro}M. This is the first study that identified an inhibitor that targets the conserved RNA-binding region of NS5 MTase, suggesting its potential as a highly effective scaffold for broad-spectrum antiviral agents against orthoflaviviruses. ImportanceDengue virus (DENV) is a vector-borne disease affecting global health, and no antiviral treatment is available for Dengue fever. DENV NS5 methyltransferase (DENV NS5 MTase) is a viral RNA capping enzyme essential for the virus replication. This study demonstrated that herbacetin (HC), a natural flavonoid, blocked the binding of GTP to NS5 MTase and inhibited the enzyme. In addition to DENV3, HC effectively inhibited the replication of DENV1,2, and 4 serotypes. The crystal structure of the DENV3 NS5 MTase in complex with HC revealed that HC binds at the cap 0 RNA binding site, which is close to the GTP binding site and thereby perturbs the spatial arrangement of GTP binding site residues. HC, as the first identified inhibitor that targets the conserved RNA-binding site of DENV3 NS5 MTase, presents a potential scaffold for the development of new antiviral drugs.

microbiology↗

bioRxiv2024

Transposable elements are prevalent vectors of transgenerational epigenetic inheritance in nature

Loss of DNA methylation over transposable elements (TEs) can affect neighboring genes and be epigenetically inherited in plants, yet the determinants and significance of this additional system of inheritance are unknown. Here, we demonstrate at thousands of TE loci across the Arabidopsis thaliana genome, that experimentally-induced hypomethylation can be transmitted transgenerationally and reveal the role of small RNAs derived from related copies in counteracting this transmission. Using data from >700 strains collected worldwide, we uncover natural hypomethylation at hundreds of the same TE loci, often situated near stress-responsive genes. Like their experimental counterparts, most natural epivariants we tested can be inherited without DNA sequence changes and are therefore bona fide epialleles, although genetic factors modulate their recurrence or persistence. Crucially, we demonstrate that TE-mediated epiallelic variation associated with differential gene expression is generally causal and may be target of selection in specific environments, thus establishing its importance in nature.

genomics↗

bioRxiv2024

Designing and bioengineering of CDRs with higher affinity against receptor-binding domain (RBD) of SARS-CoV-2 Omicron variant

The emergence of the SARS-CoV-2 Omicron variant highlights the need for innovative strategies to address evolving viral threats. This study bioengineered three nanobodies H11-H4, C5, and H3 originally targeting the Wuhan RBD, to bind more effectively to the Omicron RBD. A structure-based in silico affinity maturation pipeline was developed to enhance their binding affinities. The pipeline consists of three key steps: high-throughput in silico mutagenesis of complementarity-determining regions (CDRs), protein-protein docking for screening, and molecular dynamics (MD) simulations for assessment of the complex stability. A total of 741, 551, and 684 mutations were introduced in H11-H4, C5, and H3 nanobodies, respectively. Protein-protein docking and MD simulations shortlisted high-affinity mutants for H11-H4(6), C5(5), and H3(6). Further, recombinant production of H11-H4 mutants and Omicron RBD enabled experimental validation through Isothermal Titration Calorimetry (ITC). The H11-H4 mutants R27E, S57D, S107K, D108W, and A110I exhibited improved binding affinities with dissociation constant (KD) values ranging from [~]8.8 to [~]27 {micro}M, compared to the H11-H4 nanobody KD of [~]32 {micro}M, representing a three-fold enhancement. This study demonstrates the potential of the developed in silico affinity maturation pipeline as a rapid, cost-effective method for repurposing nanobodies, aiding the development of robust prophylactic strategies against evolving SARS-CoV-2 variants and other pathogens. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/613860v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1ea8ff7org.highwire.dtl.DTLVardef@91d567org.highwire.dtl.DTLVardef@51629corg.highwire.dtl.DTLVardef@1212752_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Normalization of Single-cell RNA-seq Data UsingPartial Least Squares with Adaptive Fuzzy Weight

Normalization of single-cell RNA-seq (scRNA-seq) is a crucial step in downstream analysis, where raw data are adjusted to correct unwanted factors that prevent the direct comparison of expression measures. scRNA-seq data exhibits a multivariate relationship between transcript-specific expression and sequencing depth that a single scale factor cannot address. A partial least squares (PLS) regression was performed to accommodate the variability of gene expression in each condition, and upper and lower quantiles with adaptive fuzzy weights were utilized to correct unwanted biases in scRNA-seq data. The present approach was compared using real and simulated datasets across various state-of-the-art performance measures.

bioinformatics↗

bioRxiv2024

Olfactory basis for essential amino acid perception during foraging in Caenorhabditis elegans

Acquisition of essential nutrients through diet is crucial for the survival of animals. Dietary odors might enable animals to forage for nutrient-rich diets. We asked if Caenorhabditis elegans, a bacterivorous nematode, uses olfactory cues to forage for essential amino acid-rich (EAA) diets. Using the native microbiome of C. elegans, we show that worms rely on olfaction to select leucine (EAA)-enriched bacteria. Using gas chromatography, we find that leucine-enriched bacteria produce isoamyl alcohol (IAA) odor in the highest abundance. Prior adaptation of worms to IAA diminishes the diet preference of worms. Several wild isolates of C. elegans display robust responses to IAA emphasizing its ecological relevance. We find that foraging for a leucine-enriched diet is mediated via the AWC olfactory neurons. Finally, we identify SNIF-1 G protein-coupled receptor in AWC neurons as a receptor for IAA and a mediator of dietary decisions in worms. Our study identifies a receptor-ligand module underpinning foraging behavior in C. elegans.

animal behavior and cognition↗

bioRxiv2024

Insights into gene conversion and crossing-over processes from long-read sequencing of human, chimpanzee and gorilla testes and sperm

Homologous recombination rearranges genetic information during meiosis to generate new combinations of variants. Recombination also causes new mutations, affects the GC content of the genome and reduces selective interference. Here, we use HiFi long-read sequencing to directly detect crossover and gene conversion events from switches between the two haplotypes along single HiFi-reads from testis tissue of humans, chimpanzees and gorillas as well as human sperm samples. Furthermore, based on DNA methylation calls, we classify the cellular origin of reads to either somatic or germline cells in the testis tissue. We identify 1692 crossovers and 1032 gene conversions in nine samples and investigate their chromosomal distribution. Crossovers are more telomeric and correlate better with recombination maps than gene conversions. We show a strong concordance between a human double-strand break map and the human samples, but not for the other species, supporting different PRDM9-programmed double-strand break loci. We estimate the average gene conversion tract lengths to be similar and very short in all three species (means 40-100 bp, fitted well by a geometric distribution) and that 95-98% of non-crossover events do not involve tracts intersecting with polymorphism and are therefore not detectable. Finally, we detect a GC bias in the gene conversion of both single and multiple SNVs and show that the GC-biased gene conversion affects SNVs flanking crossover events. This implies that gene conversion events associated with crossover events are much longer (estimated above 500 bp) than those associated with non-crossover events. Highly accurate long-read sequencing combined with the classification of reads to specific cell types provides a new, powerful way to make individual, detailed maps of gene conversion and crossovers for any species.

evolutionary biology↗

bioRxiv2024

Impaired Cortico-Amygdala Functional Connectivity Can Drive Social Behavior Deficits in Synucleinopathies

The small molecule protein -synuclein forms insoluble aggregates in a group of neurological disorders, including Parkinsons disease and dementia with Lewy bodies (DLB), which are collectively called synucleinopathies. In PD and DLB, the amygdala has been identified as a particularly susceptible region in the brain for the deposition of Lewy-like -synuclein aggregates. Though -synuclein aggregation is closely associated with neurodegeneration, there is a poor correlation between neurodegeneration in the amygdala and the clinical features of PD/DLB. We hypothesize that, prior to neurodegeneration, -synuclein aggregation disrupts functional cortical modulation of the amygdala circuits, leading to emotion dysregulation in synucleinopathies. In the present study, we combined electrophysiology, optogenetics, mouse model of synucleinopathies, and behavioral analysis to test this hypothesis. Using an -synuclein preformed fibrils (PFFs)-based mouse model of synucleinopathies, we reported dynamic changes in the levels of -synuclein pathology in the basolateral amygdala (BLA). Such dynamic changes of pathology associated with a decreased cortico-BLA connection strength prior to a significant loss of cortical axon terminals. In parallel to the reduced cortico-BLA connection, PFFs-injected mice manifested impaired social preference behavior. The impaired sociability of PFFs-injected mice could be rescued by chemogenetic stimulation of cortico-BLA inputs. Altogether, we presented a series of evidence to delineate key circuit events associated with -synuclein pathology development in the amygdala circuits. The present work highlights the necessity of a thorough investigation of functional consequences of -synuclein aggregation to advance our understand of pathophysiology of synucleinopathies and development of effective therapies.

neuroscience↗

bioRxiv2024

ATTRv-V30M Type A amyloid fibrils from the heart and nerves exhibit structural homogeneity.

ATTR amyloidosis is a systemic disease characterized by the deposition of amyloid fibrils made of transthyretin, a protein integral to transporting retinol and thyroid hormones. Transthyretin is primarily produced by the liver and circulates in blood as a tetramer. The retinal epithelium also secretes transthyretin, which is secreted to the vitreous humor of the eye. Because of mutations or aging, transthyretin can dissociate into amyloidogenic monomers triggering amyloid fibril formation. The deposition of transthyretin amyloid fibrils in the myocardium and peripheral nerves causes cardiomyopathies and neuropathies, respectively. Using cryo-electron microscopy, here we determined the structures of amyloid fibrils extracted from cardiac and nerve tissues of an ATTRv-V30M patient. We found that fibrils from both tissues share a consistent structural conformation, similar to the previously described structure of cardiac fibrils from an individual with the same genotype, but different from the fibril structure obtained from the vitreous humor. Our study hints to a uniform fibrillar architecture across different tissues within the same individual, only when the source of transthyretin is the liver. Moreover, this study provides the first description of ATTR fibrils from the nerves of a patient and enhances our understanding of the role of deposition site and protein production site in shaping the fibril structure in ATTRv-V30M amyloidosis.

biophysics↗