Biology subjects

Singh, A.

Publications and source records attributed to Singh, A..

At least 433 records · Page 24Linked to original sources

bioRxiv2022

Genetic, clinical underpinnings of subtle early brain change along Alzheimer's dimensions

Alzheimers disease (AD) is associated with heterogeneous atrophy patterns. We employed a semi-supervised clustering technique known as Surreal-GAN, through which we identified two dominant dimensions of brain atrophy in symptomatic mild cognitive impairment (MCI) and AD patients: the "diffuse-AD" (R1) dimension shows widespread brain atrophy, and the "MTL-AD" (R2) dimension displays focal medial temporal lobe (MTL) atrophy. Critically, only R2 was associated with widely known sporadic AD genetic risk factors (e.g., APOE {varepsilon}4) in MCI and AD patients at baseline. We then independently detected the presence of the two dimensions in the early stages by deploying the trained model in the general population and two cognitively unimpaired cohorts of asymptomatic participants. In the general population, genome-wide association studies found 77 genes unrelated to APOE differentially associated with R1 and R2. Functional analyses revealed that these genes were overrepresented in differentially expressed gene sets in organs beyond the brain (R1 and R2), including the heart (R1) and the pituitary gland, muscle, and kidney (R2). These genes were enriched in biological pathways implicated in dendritic cells (R2), macrophage functions (R1), and cancer (R1 and R2). Several of them were "druggable genes" for cancer (R1), inflammation (R1), cardiovascular diseases (R1), and diseases of the nervous system (R2). The longitudinal progression showed that APOE {varepsilon}4, amyloid, and tau were associated with R2 at early asymptomatic stages, but this longitudinal association occurs only at late symptomatic stages in R1. Our findings deepen our understanding of the multifaceted pathogenesis of AD beyond the brain. In early asymptomatic stages, the two dimensions are associated with diverse pathological mechanisms, including cardiovascular diseases, inflammation, and hormonal dysfunction - driven by genes different from APOE - which may collectively contribute to the early pathogenesis of AD.

bioengineering↗

bioRxiv2022

Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies reactive to complexes of platelet factor 4 and heparin. Platelets (PLTs) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations to this prothrombotic enviroment remain poorly understood. In this study, we observed that HIT patient antibodies (Abs) induce relevant changes in PLT phenotype, with the key features being increased P-Selectin expression and procoagulant phosphatidylserine (PS) externalization. Formation of procoagulant PLTs was dependent on engagement of PLT Fc-gamma-RIIA by HIT Abs and resulted in significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLTs propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLTs intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of high P-Selectin and PS levels on procoagulant PLTs was dissected. While inhibition of P-Selectin did not affect thrombus formation, the specific blockade of PS with Lactadherin prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLTs are critical mediators of prothrombotic conditions in HIT. Upregulation of cAMP with Iloprost or PS targeting specifc therapeutics could be a promising approach to prevent thromboembolic events in HIT patients. Key points- HIT immune complexes drive procoagulant platelet formation - Phosphatidylserine blockade prevents HIT antibody-induced thrombus formation

immunology↗

bioRxiv2022

Modeling cell size regulation under complex and dynamic environments

In nature, cells face changes in environmental conditions that can modify their growth rate. In these dynamic environments, recent experiments found changes in cell size regulation. Currently, there are few clues about the origin of these cell size changes. In this work, we model cell division as a stochastic process that occurs at a rate proportional to the size. We propose that this rate is zero if the cell is smaller than a minimum size. We show how this model predicts some of the properties found in cell size regulation. For example, among our predictions, we found that the mean cell size is an exponential function of the growth rate under steady conditions. We predict that cells become smaller and the way the division strategy changes during dynamic nutrient depletion. Finally, we use the model to predict cell regulation in an arbitrary complex dynamic environment.

systems biology↗

bioRxiv2022

Transiently heritable fates and quorum sensing drive early IFN-I response dynamics

Type I Interferon (IFN-I)-mediated antiviral responses are central to host defense against viral infections. Crucial is the tight and well-orchestrated control of cellular decision-making leading to the production of IFN-Is. Innovative single-cell approaches revealed that the initiation of IFN-I production is only limited to a fraction of 1-3% of the total population, both found in vitro and in vivo, which were thought to be stochastically regulated. To challenge this dogma, we addressed the influence of various host-intrinsic factors, both stochastic and epigenetic, on dictating early IFN-I responses. Hypomethylating drugs increased the percentage of responding cells. Next, with the classical Luria-Delbruck fluctuation test, we provided evidence that the fate of becoming a responding cell is transiently heritable. Finally, while studying varying cell-densities, we substantiated an important role for quorum sensing, which was verified by mathematical modeling. Together, this systems immunology approach opens up new avenues to progress the fundamental understanding of cellular decision-making during early IFN-I responses, which can be translated to other (immune) signaling systems, and ultimately will improve IFN-I based immune therapies.

immunology↗

bioRxiv2022

Effect of 15 days hindlimb unloading on baroreflex sensitivity, Hear rate variability and Carotid artery structure in rats

IntroductionIn space, cephalad shift of body fluid equalizes the blood pressure throughout the body leading to various cardiovascular disturbances in space and upon return on earth. Effects of microgravity on humans can be simulated on earth by hindlimb unloading (HU) rat models l. This study was planned to examine the Effect of 15 days hindlimb unloading on baroreflex sensitivity, Herat rate variability (HRV) and carotid artery structure in rats. Material and methods18 male Wistar rats weighing 250-300 gm were randomly divided into control and hindlimb unloading groups and housed in separate cages for 15 days. Body weight, food and water intake was measured every day. The pelvic suspension method was used to simulate effects of microgravity. To record HRV, ECG was recorded in lead II configuration on the 15th day. Then blood pressure was recorded directly from the carotid artery. Linear regression of arterial pressure and RR interval used to derive the Baroreflex sensitivity. Vessel wall thickness, intimal thickness, smooth muscle cell layer thickness & luminal diameter were compared after H&E staining. Ultrathin sectioning and counter-staining was done for electron microscopy and Changes in endothelial thickness, elastic lamina, and deeper artery structures were observed. Statistical analysis was done using Graphpad Prism (version 9.0). Unpaired t-test, Mann Whitney and 2 way ANOVA were used according to groups and distribution of parameters. ResultsWe found significant differences in Food intake (p {square}0.001), Body weight (p {square}0.001), Heart rate (p=0.031), BRS (p= 0.026), R-R interval (p=0.0246), heart rate variability parameters {SDSD (p=0.0001), RMSD (p= 0.0001), VLF(p=0.001),HF (p=0.0054), LF/HF Ratio (p= 0.0001)}. We did not found significant changes in Water intake, MAP, SBP, DBP, Vessel wall thickness, Luminal Diameter, Intimal thickness, Smooth muscle thickness, in H&E staining and in electron microscopy. ConclusionChronic microgravity simulation using pelvic suspension showed a decrease in BRS after 15 days of HU; however 15 days of HU was not insufficient to produce any structural changes in the carotid artery. BRS changes were accompanied by change in vascular vasoconstriction and vasodilation properties which were due to alteration in the sympathetic and parasympathetic outflow.

physiology↗

bioRxiv2022

Violation of rationality in shoaling decision making in zebrafish (Danio rerio)

The decoy effect, a bias in choice between two options when a third, inferior option is introduced, has been observed across various organisms, from slime molds to humans. In this study, we investigated whether zebrafish (Danio rerio), a widely used biological model organism, exhibit the decoy effect in their shoaling group choices, specifically examining this effect by varying only shoal size. Using spatial trajectory analysis of freely swimming zebrafish interacting with conspecifics in adjacent display tanks, we tested how shoaling decisions varied between dichotomous (two-option) and trichotomous (three-option) choice sets. Our experiments compared preferences for 4 versus 2 and 6 versus 3 shoal sizes, with a single fish serving as a decoy in the trichotomous sets. The results revealed sex-specific differences in the decoy effect: males exhibited a shift in preference only in the Trichotomous-first condition, where prior exposure to the decoy led to a significant preference for the larger shoal. In contrast, females displayed the decoy effect exclusively in the Dichotomous-first condition, shifting from a significant preference for the larger shoal to showing no clear preference in the presence of the decoy. Notably, our findings demonstrate that the decoy effect can occur even when studied unidimensionally, with both sex and the order of presentation influencing zebrafish shoaling preferences. These results offer new insights into decision-making processes and highlight the importance of considering order effects in studies of choice behavior, with potential methodological implications for research in other species.

animal behavior and cognition↗

bioRxiv2022

Enhancer Reprogramming in Melanoma Immune Checkpoint Therapy Resistance

Immune checkpoint blockade (ICB) therapy has improved long-term survival for patients with advanced melanoma. However, there is critical need to identify potential biomarkers of response and actionable strategies to improve response rates. Through generation and analysis of 148 chromatin modification maps for 36 melanoma samples from patients treated with anti-PD- 1, we identified significant enrichment of active enhancer states in non-responders at baseline. Analysis of an independent cohort of 20 samples identified a set of 437 enhancers that predicted response to anti-PD-1 therapy (Area Under the Curve of 0.8417). The activated non-responder enhancers marked a group of key regulators of several pathways in melanoma cells (including c- MET, TGF{beta}, EMT and AKT) that are known to mediate resistance to ICB therapy and several checkpoint receptors in T cells. Epigenetic editing experiments implicated involvement of c-MET enhancers in the modulation of immune response. Finally, inhibition of enhancers and repression of these pathways using bromodomain inhibitors along with anti-PD-1 therapy significantly decreased melanoma tumor burden and increased T-cell infiltration. Together, these findings identify a potential enhancer-based biomarker of resistance to anti-PD-1 and suggest enhancer blockade in combination with ICB as a potential strategy to improve responses.

cancer biology↗

bioRxiv2022

Myoglobin promotes cardiomyocyte differentiation through oxidatively modulating the Hippo Kinase Pathway

BackgroundWhile cardiomyocytes undergo terminal differentiation postnatally and rarely re-enter the cell cycle, the endogenous mechanisms that propagate differentiation and prevent de-differentiation remain unclear. The monomeric heme protein myoglobin, which stores oxygen and regulates reactive oxygen/nitrogen species balance in the heart, increases in expression by over 50% during cardiomyocyte differentiation. Though myoglobin deletion without significant compensation is embryonic lethal in mice, a role for the protein in regulating cardiomyocyte differentiation has not been tested. We hypothesized that myoglobin expression is required for cardiomyocyte differentiation and the loss of myoglobin enables de-differentiation. MethodsMyoglobin was genetically silenced in HL-1, H9C2 cells, and neonatal rat ventricular cardiomyocytes (NRVM) to examine myoglobin-dependent effects on differentiation, proliferation, and Hippo pathway signaling. A zebrafish model of Mb depletion was made using CRISPR-Cas9 to test the effect of myoglobin depletion on cardiac regeneration after apical resection injury in vivo. ResultsMyoglobin deletion in cultured cell lines and NRVM decreased the gene expression of cardiomyocyte differentiation markers (troponin, myosin light chain, and myosin heavy chain), upregulated markers of dedifferentiation (runx1 and dab2) and stimulated cell proliferation. Mechanistically, we show that the heme prosthetic group of myoglobin catalyzes the oxidation of the Hippo pathway kinase LATS1, which activates the enzyme to phosphorylate the downstream Yes-associated protein (YAP) transcription factor, which prevents its transcriptional activity. Thus, the loss of myoglobin results in the de-phosphorylation and nuclear translocation of YAP, which propagates proliferation and fetal gene expression. In vivo, myoglobin-deficient zebrafish hearts recapitulated the increase in YAP signaling and showed accelerated regeneration at 20 days post apical injury. ConclusionWe a novel role for myoglobin as an endogenous driver of cardiomyocyte differentiation, and a regulator of the Hippo pathway. These findings suggest myoglobin as a potential target for strategies to enhance cardiac development and improve cardiac repair and regeneration.

cell biology↗

bioRxiv2022

Diffusion controls local versus dispersed inheritance of histones during replication and shapes epigenomic architecture

The dynamics of inheritance of histones and their associated modifications across cell divisions can have major consequences on maintenance of the cellular epigenomic state. Recent experiments contradict the long-held notion that histone inheritance during replication is always local, suggesting that active and repressed regions of the genome exhibit fundamentally different histone dynamics independent of transcription-coupled turnover. Here we develop a stochastic model of histone dynamics at the replication fork and demonstrate that differential diffusivity of histones in active versus repressed chromatin is sufficient to quantitatively explain these recent experiments. Further, we use the model to predict patterns in histone mark similarity between pairs of genomic loci that should be developed as a result of diffusion, but not from PRC2 mediated mark spreading or transcriptional processes. Interestingly, using a combination of CHIP-seq, replication timing and Hi-C datasets we demonstrate that all the computationally predicted patterns are consistently observed for both active and repressive histone marks in two different cell lines. Our results suggest that in contrast to current models that posit histone transfer exclusively between parental and daughter DNA strands, dislodged histones in euchromatin and facultative heterochromatin diffuse within larger "Diffusion-Accessible-Domains" (DADs), leading to redistribution of epigenetic marks both within and across chromosomes. Preservation of the epigenomic state across cell divisions therefore might be achieved not by passing on strict positional information of histone marks, but by maintaining the marks in somewhat larger DADs of the genome.

biophysics↗

bioRxiv2022

Predicting Dementia Severity by Merging Anatomical and Diffusion MRI with Deep 3D Convolutional Neural Networks

Machine learning methods have been used for over a decade for staging and subtyping a variety of brain diseases, offering fast and objective methods to classify neurodegenerative diseases such as Alzheimers disease (AD). Deep learning models based on convolutional neural networks (CNNs) have also been used to infer dementia severity and predict future clinical decline. Most CNN-based deep learning models use T1-weighted brain MRI scans to identify predictive features for these tasks. In contrast, we examine the added value of diffusion-weighted MRI (dMRI) - a variant of MRI, sensitive to microstructural tissue properties - as an additional input in CNN-based models of dementia severity. dMRI is sensitive to microstructural brain abnormalities not evident on standard anatomical MRI. By training CNNs on combined anatomical and diffusion MRI, we hypothesize that we could boost performance when predicting widely-used clinical assessments of dementia severity, such as individuals scores on the ADAS11, ADAS13, and MMSE (mini-mental state exam) clinical scales. For benchmarking, we evaluate CNNs that use T1-weighted MRI and dMRI to estimate "brain age" - the task of predicting a persons chronological age from their neuroimaging data. To assess which dMRI-derived maps were most beneficial, we computed DWI-derived diffusion tensor imaging (DTI) maps of mean and radial diffusivity (MD/RD), axial diffusivity (AD) and fractional anisotropy (FA) for 1198 elderly subjects (age: 74.35 +/- 7.74 yrs.; 600 F/598 M, with a distribution of 636 CN/421 MCI/141 AD) from the Alzheimers Disease Neuroimaging Initiative (ADNI). We tested both 2D Slice CNN and 3D CNN neural network models for the above predictive tasks. Our results suggest that for at least some deep learning architectures, diffusion-weighted MRI may enhance performance for several AD-relevant deep learning tasks relative to using T1-weighted images alone.

neuroscience↗

bioRxiv2022

UBA52 is crucial in HSP90 ubiquitylation and neurodegenerative signaling during early phase of Parkinson disease

Protein aggregation is one of the major pathological events in age-related Parkinsons disease (PD) pathology, predominantly regulated by the ubiquitin-proteasome system (UPS). UPS essentially requires core component ubiquitin however, its role in PD pathology is obscure. This study aimed to investigate the role of ubiquitin encoding genes in the early phase of PD pathology. Wild-type human Myc--synuclein transfected neurons, -synuclein-PFFs treated cells, rotenone-induced sporadic models of PD and SNCA C57BL/6J-Tg (Th-SNCA*A30P*A53T)39 Eric/J transgenic mice showed downregulated level of UBA52 in conjunction with significant downregulation of tyrosine hydroxylase (TH) and neuronal death. In silico predictions, mass spectrometric analysis and co-immunoprecipitation findings suggested strong interaction of UBA52 with -synuclein, HSP90 and E3-ubiquitin ligase CHIP, besides its co-localization with -synuclein in the mitochondrion. Next, in vitro ubiquitylation assay indicated an imperative requirement of the lysine-63 residue of UBA52 in CHIP-mediated HSP90 ubiquitylation. Myc-UBA52 expressed neurons exhibited the downregulated -synuclein protein abundance with increased TH and restored proteasome activity during the diseased condition. Furthermore, Myc-UBA52 expression inhibited the augmented HSP90 protein level along with its various client proteins, HSP75 (homologue of HSP90 in mitochondrion) and ER stress-related markers during early PD. Taken together, data highlights the critical role of UBA52 in HSP90 ubiquitylation in parallel to its potential contribution to the modulation of various disease-related neurodegenerative signaling targets during the early phase of PD pathology.

neuroscience↗

bioRxiv2022

Labeled lines for fat and sugar reward combine to promote overeating

Food is a powerful natural reinforcer that guides feeding decisions. The vagus nerve conveys internal sensory information from the gut to the brain about nutritional value; however, the cellular and molecular basis of macronutrient-specific reward circuits are poorly understood. Here, we monitor in vivo calcium dynamics to provide direct evidence of independent vagal sensing pathways for detection of dietary fats and sugars. Using activity-dependent genetic capture of nutrient-specific vagal neurons activated in response gut infusions, we demonstrate the existence of separate hard-wired reward circuits for fat and sugar that are necessary and sufficient for nutrient-specific reinforcement. Even when controlling for calories, combined activation of fat and sugar circuits increases overeating compared to fat or sugar alone. This work provides new insight on the complex sensory circuitry that mediates motivated behavior and suggests that a subconscious internal drive to consume obesogenic diets (e.g., those high in both fat and sugar) may impede conscious dieting efforts.

neuroscience↗

bioRxiv2022

A Genetic Variant of Fatty Acid Amide Hydrolase (FAAH) Exacerbates Hormone- Mediated Orexigenic Feeding in Mice

Fatty acid amide hydrolase (FAAH) degrades the endocannabinoid anandamide. A polymorphism in FAAH (FAAH C385A) reduces FAAH expression, increases anandamide levels, and increases the risk of obesity. Nevertheless, some studies have found no association between FAAH C385A and obesity. We investigated whether the environmental context governs the impact of FAAH C385A on metabolic outcomes. Using a C385A knock-in mouse model, we found that under basal conditions, there was no effect of the FAAH C385A polymorphism on body weight or composition. By contrast, FAAH A/A mice are more susceptible to glucocorticoid-induced hyperphagia, weight gain, and activation of hypothalamic AMPK. AMPK inhibition occluded the amplified hyperphagic response to glucocorticoids in FAAH A/A mice. FAAH knockdown exclusively in AgRP neurons mimicked the exaggerated feeding response of FAAH A/A mice to glucocorticoids. FAAH A/A mice likewise presented exaggerated orexigenic responses to ghrelin, while FAAH knockdown in AgRP neurons blunted leptin anorectic responses. Together, the FAAH A/A genotype amplifies orexigenic responses and decreases anorexigenic responses, providing a putative mechanism explaining the diverging human findings.

neuroscience↗

bioRxiv2022

An investigation of the sex-specific genetic architecture of fitness in Drosophila melanogaster

In dioecious populations, the sexes employ divergent reproductive strategies to maximize fitness and, as a result, genetic variants can affect fitness differently in males and females. Moreover, recent studies have highlighted an important role of the mating environment in shaping the strength and direction of sex-specific selection. Here, we measure adult fitness for each sex of 357 lines from the Drosophila Synthetic Population Resource (DSPR) in two different mating environments. We analyze the data using three different approaches to gain insight into the sex-specific genetic architecture for fitness: classical quantitative genetics, genomic associations, and a mutational burden approach. The quantitative genetics analysis finds that, on average segregating genetic variation in this population has concordant fitness effects both across the sexes and across mating environments. We do not find specific genomic regions with strong associations with either sexually antagonistic (SA) or sexually concordant (SC) fitness effects, yet there is modest evidence of an excess of genomic regions with weak associations, both with SA and SC fitness effects. Our examination of mutational burden indicates stronger selection against indels and loss-of-function variants in females than males.

evolutionary biology↗

bioRxiv2022

Dynamics and consequences of the HTLV-1 proviral plus-strand burst

Expression of the transcriptional transactivator protein Tax, encoded on the proviral plus-strand of human T-cell leukaemia virus type 1 (HTLV-1), is crucial for the replication of the virus, but Tax-expressing cells are rarely detected in fresh blood ex vivo. The dynamics and consequences of the proviral plus-strand transcriptional burst remain insufficiently characterised. We combined time-lapse live-cell imaging, single-cell tracking and mathematical modelling to study the dynamics of Tax expression at single-cell resolution in two naturally-infected T-cell clones transduced with a short-lived enhanced green fluorescent protein (d2EGFP) Tax reporter system. Five different patterns of Tax expression were observed during the 30-hour observation period; the distribution of these patterns differed between the two clones. The mean duration of Tax expression in the two clones was 94 and 417 hours respectively, estimated from mathematical modelling of the experimental data. Tax expression was associated with decreased proliferation, increased apoptosis, enhanced activation of the DNA damage response pathways, and slower cell-cycle progression. Longer-term follow-up (14 days) revealed an increase in the proportion of proliferating cells and a decrease in the fraction of apoptotic cells as the cells ceased Tax expression, resulting in a greater net expansion of the initially Tax-positive population. Time-lapse live-cell imaging showed enhanced cell-to-cell adhesion among Tax-expressing cells, and decreased cell motility of Tax-expressing cells at the single-cell level. The results demonstrate the within-clone and between-clone heterogeneity in the dynamics and patterns of HTLV-1 plus-strand transcriptional bursts and the balance of positive and negative consequences of the burst for the host cell. Author SummaryHuman T-cell leukaemia virus type 1 (HTLV-1) causes disabling or fatal diseases in up to 10% of the infected individuals. The expression of viral protein Tax is essential to cause new infections and contributes to HTLV-1-associated diseases. The proviral plus-strand, which encodes Tax, is expressed in intense intermittent bursts. However, the kinetics of Tax expression and its short and longer-term impact on the infected cell are not well understood. We combined live-cell imaging and mathematical modelling to study Tax expression kinetics in two naturally-infected T-cell clones. Single-cell analysis showed five patterns of Tax expression, with most Tax-positive cells expressing continuously during the 30-hour imaging. The average duration of Tax expression in the two clones was 94 and 417 hours respectively, by mathematical modelling. Tax expression correlated with decreased proliferation, increased apoptosis, enhanced activation of DNA damage response pathways and delayed progression through the cell-cycle. Extended observation showed an increase in the proportion of proliferating cells and a decrease in the percentage of apoptotic cells as cells ceased Tax expression, resulting in a greater net growth of the originally Tax-positive population. Tax-expressing cells also formed cell clumps and showed reduced cell movement. This study highlights prolonged Tax expression and its short and longer-term impact on naturally-infected cells.

microbiology↗

bioRxiv2022

The role of division stochasticity on the robustness of bacterial size dynamics

Variables of bacterial division such as size at birth, growth rate, division time, and the position of the septal ring, all vary from cell to cell. Currently, it is unknown how these random fluctuations can combine to produce a robust mechanism of homeostasis. To address this question, we studied the dynamics of the cell division process from both experimental and theoretical perspectives. Our model predicts robustness in division times as sustained oscillations in metrics of the cell size distribution, such as the mean, variability, and the cell size autocorrelation function. These oscillations do not get damped, even considering stochasticity in division timing and the cell size at the beginning of the experiment. Damping appears just after inducing stochasticity in either the septum position or the growth rate. We compare the predictions of the full model with the size dynamics of E. coli bacteria growing in minimal media using either glucose or glycerol as carbon sources. We observe that growth in poorer media increases the noise in both partitioning position and growth rate. This additional noise results in oscillations with more damping. Although intracellular noise is known as a source of phenotypic variation, our results show that it can play a similar but subtler role in maintaining population-level homeostasis by causing rapid desynchronization of cell cycles..

systems biology↗

bioRxiv2022

HAT: Haplotype Assembly Tool using short and error-prone long reads

MotivationHaplotypes are the set of alleles cooccurring on a single chromosome and inherited together to the next generation. Because a monoploid reference genome loses this co-occurrence information, its usability is limited to associate phenotypes with allelic combinations of genotypes. Therefore, methods to reconstruct the complete haplotypes from DNA sequencing data are crucial. Recently, several attempts have been made at haplotype reconstructions, but significant limitations remain. High-quality continuous haplotypes cannot be created reliably, particularly when there are few differences between the homologous chromosomes. ResultsHere, we introduce HAT, a haplotype assembly tool that exploits short and long reads along with a reference genome to reconstruct haplotypes. HAT tries to take advantage of the accuracy of short reads and the length of the long reads to reconstruct haplotypes. We tested HAT on the aneuploid yeast strain Saccharomyces pastorianus CBS1483 and multiple simulated polyploid data sets of the same strain, showing that it outperforms existing tools. Availabilityhttps://github.com/AbeelLab/hat/ Contactt.abeel@tudelft.nl

bioinformatics↗

bioRxiv2022

GTPase-dependent cyclic flexibility transitions drive the two-component EEA1-Rab5 molecular motor

The recognition of vesicles by their correct target compartment depends on the pairing of small GTPases with effector proteins (1-3). Whereas ATPases can cyclically convert the free energy of ATP hydrolysis into mechanical work, the function of small GTPases is predominantly associated with signal transduction processes, and their role in mechano-transduction is less established (4-6). However, binding of the GTPase Rab5 to the long coiled-coil tethering protein EEA1 on an early endosome induces a rigidity transition resulting in a large conformational change in EEA1 from a rigid and extended to a flexible and collapsed state. This entropic collapse of EEA1 gives rise to an effective force that can pull tethered membranes closer (7). It currently remains unclear if EEA1 can return from the collapsed to the extended conformation without the aid of chaperones. Here, we use fluorescence correlation spectroscopy to reveal that EEA1 in bulk solution can undergo multiple flexibility transition cycles that are associated with the binding and release of Rab5(GTP) and Rab5(GDP). Using semi-flexible polymer theory we provide evidence that the cyclic transitioning of Rab5-EEA1 between extended and collapsed conformations is driven by the energetics of Rab5 binding/unbinding and GTP hydrolysis. Cyclic flexibility transitions represent a complete mechanical work cycle that is able to perform up to 20 kBT of mechanical work per cycle, against an opposing force. Hence, Rab5 and EEA1 constitute a two-component molecular motor driven by the chemical energy derived from GTP hydrolysis by Rab5. We conclude that coiled-coil tethering proteins and their small GTPase partners can have active mechanical roles in membrane trafficking.

biophysics↗