Biology subjects

Singh, A.

Publications and source records attributed to Singh, A..

At least 289 records · Page 16Linked to original sources

bioRxiv2024

Methemoglobin aggregation is modulated by the anti-sickling drug voxelotor

Sickle cell disease is caused by a mutation in the beta subunit of hemoglobin (HbSS) that drives Hb fiber formation when the protein is in the deoxygenated (tense, T) state. The drug voxelotor was recently approved to treat sickle cell disease by preventing HbSS fiber formation. Voxelotor acts as an allosteric inhibitor of polymerization by maintaining the HbSS protein in the relaxed (R) conformation, limiting polymerization of T-state fibers. Normal blood cells contain small amounts of natural Hb fibers and a few percent of the Fe3+ ferric form, metHb, incapable of binding oxygen. Although the drug Voxelotor is now in use, the effect of the drug on the oxidized metHb state has not been reported. Here we assessed the influence of voxelotor on normal human metHb. We compared the aggregation of metHb at two pH values (5.5 and 7.1). MetHb is known to form organized fiber structures at or below pH 5.5. We find that voxelotor significantly enhances fiber formation of metHb R-state at pH 5.5, consistent with the mode of action for this drug in maintaining the Hb R conformation. The opposite effect is observed at physiological pH values. Voxelotor significantly decreases the rate of metHb aggregate formation at pH 7.1 but did not affect protein stability. Notably, drug binding drives metHb into novel spherical particles with a morphology never seen before for Hb. The formation of these particles should be considered in patients being treated for sickle cell disease with voxelotor. WHY IT MATTERSVoxelotor is an FDA-approved drug for sickle cell anemia, known to prevent hemoglobin fiber formation. Here, we investigate its effect on methemoglobin, the form of hemoglobin in which iron takes on the ferric Fe3+ state. Our study examines voxelotors impact on methemoglobin aggregation and stability. At pH 7.1, we found voxelotor to have an effect on methemoglobin solubility as evidenced by the formation of novel methemoglobin spherical structures. We observe that voxelotor significantly increases methemoglobin fiber formation at pH 5.5 but, notably, reduces methemoglobin aggregation at physiological pH levels. Minimal impact on methemoglobin thermodynamic stability is noted. These findings suggest voxelotors potential therapeutic efficacy for various hemoglobinopathies, including conditions characterized by Heinz body formation.

biophysics↗

bioRxiv2024

Promoting Human Intestinal Organoid Formation and Stimulation Using Piezoelectric Nanofiber Matrices

Human organoid model systems have changed the landscape of developmental biology and basic science. They serve as a great tool for human specific interrogation. In order to advance our organoid technology, we aimed to test the compatibility of a piezoelectric material with organoid generation, because it will create a new platform with the potential for sensing and actuating organoids in physiologically relevant ways. We differentiated human pluripotent stem cells into spheroids following the traditional human intestinal organoid (HIO) protocol atop a piezoelectric nanofiber scaffold. We observed that exposure to the biocompatible piezoelectric nanofibers promoted spheroid morphology three days sooner than with the conventional methodology. At day 28 of culture, HIOs grown on the scaffold appeared similar. Both groups were readily transplantable and developed well-organized laminated structures. Graft sizes between groups were similar. Upon characterizing the tissue further, we found no detrimental effects of the piezoelectric nanofibers on intestinal patterning or maturation. Furthermore, to test the practical feasibility of the material, HIOs were also matured on the nanofiber scaffolds and treated with ultrasound, which lead to increased cellular proliferation which is critical for organoid development and tissue maintenance. This study establishes a proof of concept for integrating piezoelectric materials as a customizable platform for on-demand electrical stimulation of cells using remote ultrasonic waveforms in regenerative medicine.

bioengineering↗

bioRxiv2024

Biomolecular Condensates Can Enhance Pathological RNA Clustering

Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant, and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures, with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which accompanies a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.

biophysics↗

bioRxiv2024

Leveraging the transient statistics of quantal content to infer neuronal synaptic transmission

Quantal parameters of synapses are fundamental for the temporal dynamics of neurotransmitter release, forming the basis of interneuronal communication. We propose a class of models that capture the stochastic dynamics of quantal content (QC) - the number of SV fusion events per action potential (AP). Considering the probabilistic and time-varying nature of SV docking, undocking, and AP-triggered fusion, we derive an exact statistical distribution for the QC over time. Analyzing this distribution at steady-state and its associated autocorrelation function, we show that QC fluctuation statistics can be leveraged for inferring key presynaptic parameters, such as the probability of SV fusion (release probability), and SV replenishment at empty docking sites (refilling probability). Our model predictions are tested with electrophysiological data obtained from 50-Hz stimulation of auditory MNTB-LSO synapses in brainstem slices of juvenile mice. Our results show that while synaptic depression can be explained by low and constant refilling/release probabilities, this scenario is inconsistent with the statistics of the electrophysiological data that show a low QC Fano factor and almost uncorrelated successive QCs. Our systematic analysis yields a model that couples a high release probability to a time-varying refilling probability to explain both the synaptic depression and its associated statistical fluctuations. In summary, we provide a general approach that exploits stochastic signatures in QCs to infer neurotransmission-regulating processes that are indistinguishable from just analyzing averaged synaptic responses.

neuroscience↗

bioRxiv2024

HIV-1 neutralizing antibodies in SHIV-infected macaques recapitulate structurally divergent modes of human V2 apex recognition with a single D gene

Broadly neutralizing antibodies targeting the V2 apex of the HIV-1 envelope trimer are among the most common specificities elicited in HIV-1-infected humans and simian-human immunodeficiency virus (SHIV)-infected macaques. To gain insight into the prevalent induction of these antibodies, we isolated and characterized 11 V2 apex-directed neutralizing antibody lineages from SHIV-infected rhesus macaques. Remarkably, all SHIV-induced V2 apex lineages were derived from reading frame two of the rhesus DH3-15*01 gene. Cryo-EM structures of envelope trimers in complex with antibodies from nine rhesus lineages revealed modes of recognition that mimicked three canonical human V2 apex-recognition modes. Notably, amino acids encoded by DH3-15*01 played divergent structural roles, inserting into a hole at the trimer apex, H-bonding to an exposed strand, or forming part of a loop scaffold. Overall, we identify a DH3-15*01-signature for rhesus V2 apex broadly neutralizing antibodies and show that highly selected genetic elements can play multiple roles in antigen recognition. HighlightsO_LIIsolated 11 V2 apex-targeted HIV-neutralizing lineages from 10 SHIV-infected Indian-origin rhesus macaques C_LIO_LICryo-EM structures of Fab-Env complexes for nine rhesus lineages reveal modes of recognition that mimic three modes of human V2 apex antibody recognition C_LIO_LIAll SHIV-elicited V2 apex lineages, including two others previously published, derive from the same DH3-15*01 gene utilizing reading frame two C_LIO_LIThe DH3-15*01 gene in reading frame two provides a necessary, but not sufficient, signature for V2 apex-directed broadly neutralizing antibodies C_LIO_LIStructural roles played by DH3-15*01-encoded amino acids differed substantially in different lineages, even for those with the same recognition mode C_LIO_LIPropose that the anionic, aromatic, and extended character of DH3-15*01 in reading frame two provides a selective advantage for V2 apex recognition compared to B cells derived from other D genes in the naive rhesus repertoire C_LIO_LIDemonstrate that highly selected genetic elements can play multiple roles in antigen recognition, providing a structural means to enhance recognition diversity C_LI

immunology↗

bioRxiv2024

Systemic and targeted activation of Nrf2 reverses doxorubicin-induced cognitive impairments and sensorimotor deficits in mice

While cancer survivorship has increased due to advances in treatments, chemotherapy often carries long-lived neurotoxic side effects which reduce quality of life. Commonly affected domains include memory, executive function, attention, processing speed and sensorimotor function, colloquially known as chemotherapy-induced cognitive impairment (CICI) or "chemobrain". Oxidative stress and neuroimmune signaling in the brain have been mechanistically linked to the deleterious effects of chemotherapy on cognition and sensorimotor function. With this in mind, we tested if activation of the master regulator of antioxidant response nuclear factor E2-related factor 2 (Nrf2) alleviates cognitive and sensorimotor impairments induced by doxorubicin. The FDA-approved systemic Nrf2 activator, diroximel fumarate (DRF) was used, along with our recently developed prodrug 1c which has the advantage of specifically releasing monomethyl fumarate at sites of oxidative stress. DRF and 1c both reversed doxorubicin-induced deficits in executive function, spatial and working memory, as well as decrements in fine motor coordination and grip strength, across both male and female mice. Both treatments reversed doxorubicin-induced loss of synaptic proteins and microglia phenotypic transition in the hippocampus. Doxorubicin-induced myelin damage in the corpus callosum was reversed by both Nrf2 activators. These results demonstrate the therapeutic potential of Nrf2 activators to reverse doxorubicin-induced cognitive impairments, motor incoordination, and associated structural and phenotypic changes in the brain. The localized release of monomethyl fumarate by 1c has the potential to diminish unwanted effects of fumarates while retaining efficacy.

neuroscience↗

bioRxiv2024

Bioinformatics analysis of the potential biomarkers of Multiple Sclerosis and Guillain-Barre syndrome

Recent research emphasizes the intricate interplay of genetics and epigenetics in neurological disorders, notably Multiple Sclerosis (MS) and Guillain-Barre Syndrome (GBS), both of which exhibit cardiovascular dysregulation, with GBS often featuring serious bradyarrhythmias requiring prompt recognition and treatment. While cardiovascular autonomic dysfunction in MS is typically less severe, orthostatic intolerance affects around half of MS patients. Their distinction lies in their autoimmune responses, MS is an autoimmune disease affecting the central nervous system, causes demyelination and axon damage, leading to cognitive, ocular, and musculoskeletal dysfunction. In contrast, GBS primarily affects the peripheral nervous system, resulting in paralysis and respiratory complications. Despite their differences, both diseases share environmental risk factors such as viral infections and Vitamin D deficiency. This study aims to explore shared gene expression pathways, functional annotations, and molecular pathways between MS and GBS to enhance diagnostics, pathogenesis understanding, and treatment strategies through molecular analysis techniques. Through the gene expression analysis, five significant genes were found UTS2, TNFSF10, GBP1, VCAN, FOS. Results shows that Common DEGs are linked to apoptosis, bacterial infections, and atherosclerosis. Molecular docking analysis suggests Aflatoxin B1 as a potential therapeutic compound due to its high binding affinity with common differentially expressed proteins.

bioinformatics↗

bioRxiv2024

Programmable soft DNA hydrogels stimulate cellular endocytic pathways and proliferation

Hydrogels are pivotal in tissue engineering, regenerative medicine, and drug delivery applications. DNA molecules stand out among various biomaterials due to their unparalleled precision, programmability, and customization. In this study, we introduce a palate of novel cellular scaffolding platforms made of pure DNA-based hydrogel systems while improving the shortcomings of the existing platforms. DNA strands can form complex supramolecular branched structures essential for designing novel functional materials by its precise sequence-based self-assembly. These unique geometric scaffolds offer a soft, cushiony platform, ideal for culturing cells to mimic the complex native in vivo environments better. Each hydrogel comprises repeating units of branched DNA supramolecular structures, each possessing a distinct number of branching arms. The epithelial cells grown over these hydrogels show dynamic changes at multiple levels, from morphology to protein expression patterns, enhanced membrane traffic, and proliferation. The DNA hydrogels explored here are mechanically weak and soft and thus appropriate for applications in cell biology. This research lays the groundwork for developing a DNA hydrogel system with a higher dynamic range of stiffness, which will open exciting avenues for tissue engineering and beyond. Graphical abstract illustrating diverse branched DNA supramolecular architectures forming DNA hydrogels of various geometric profiles, each put to use in the cell culture applications. O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/595930v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1a3d4cborg.highwire.dtl.DTLVardef@e771cforg.highwire.dtl.DTLVardef@e0d5b4org.highwire.dtl.DTLVardef@ee42d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Self-assembled DNA-collagen bioactive scaffolds promote cellular uptake and neuronal differentiation

Different modalities of DNA-Collagen complexes have been utilized primarily for gene delivery studies. However, very few studies have investigated the potential of these complexes as bioactive scaffolds. Further, no studies have characterized the DNA-Collagen complex formed from the interaction of self-assembled DNA macrostructure and collagen. Towards this investigation, we report herein the fabrication of novel bioactive scaffolds formed from the interaction of sequence-specific, self-assembled DNA macrostructure and collagen type I. Varying molar ratios of DNA and collagen resulted in highly intertwined fibrous scaffolds with different fibrillar thicknesses. The formed scaffolds were biocompatible and presented as a soft matrix for cellular growth and proliferation. Cells cultured on DNA/Collagen scaffolds promoted enhanced cellular uptake of transferrin, and the potential of DNA/Collagen scaffolds to induce neuronal cell differentiation was further investigated. The DNA/Collagen scaffolds promoted neuronal differentiation of precursor cells with extensive neurite growth in comparison to control groups. These novel, self-assembled DNA/Collagen scaffolds could serve as a platform for the development of various bioactive scaffolds with potential applications in neuroscience, drug delivery, tissue engineering, and in vitro cell culture. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=137 HEIGHT=200 SRC="FIGDIR/small/595848v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@26e16aorg.highwire.dtl.DTLVardef@991d35org.highwire.dtl.DTLVardef@4c8909org.highwire.dtl.DTLVardef@b8ba55_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Gram-positive bacteria are primed for surviving lethal doses of antibiotics and chemical stress

Antibiotic resistance kills millions worldwide yearly. However, a major contributor to recurrent infections lies in a small fraction of bacterial cells, known as persisters. These cells are not inherently antibiotic-resistant, yet they lead to increased antibiotic usage, raising the risk of developing resistant progenies. In a bacterial population, individual cells exhibit considerable fluctuations in their gene expression levels despite being cultivated under identical, stable conditions. This variability in cell-to-cell characteristics (phenotypic diversity) within an isogenic population enables persister cells to withstand antibiotic exposure by entering a non-dividing state. We recently showed the existence of "primed cells" in E. coli. Primed cells are dividing cells prepared for antibiotic stress before encountering it and are more prone to form persisters. They also pass their "prepared state" down for several generations through epigenetic memory. Here, we show that primed cells are common among distant bacterial lineages, allowing for survival against antibiotics and other chemical stress, and form in different growth phases. They are also responsible for increased persister levels in transition and stationary phases compared to the log phase. We tested and showed that the Gram-positive bacterium Bacillus megaterium, evolutionarily very distant from E. coli, forms primed cells and has a transient epigenetic memory that is maintained for 7 generations or more. We showed this using ciprofloxacin and the non-antibiotic chemical stress fluoride. It is well established that persister levels are higher in the stationary phase than in the log phase, and B. megaterium persisters levels are nearly identical from the early to late-log phase but are [~]2-fold and [~]4-fold higher in the transition and stationary phase, respectively. It was previously proposed that there are two distinct types of persisters: Type II forms in the log phase, while Type I forms in the stationary phase. However, we show that primed cells lead to increased persisters in the transition and stationary phase and found no evidence of Type I or II persisters with distant phenotypes. Overall, we have provided substantial evidence of the importance of primed cells and their transitory epigenetic memories to surviving stress.

microbiology↗

bioRxiv2024

Molecular Sequestration Enhances Precision in Timing of Intracellular Events

Expressed gene products often interact ubiquitously with binding sites at nucleic acids and macromolecular complexes, known as decoys. Binding of transcription factors (TFs) to decoys is shown to be an indirect but crucial way to control the dynamics and stochasticity in gene regulation. Here, we explore how such decoys impact the timing of intracellular events, as captured by the time taken for the levels of a given TF to reach a critical threshold level, known as first passage time (FPT). As binding introduces nonlinearity, an exact mathematical analysis is challenging. However, assuming quasi-static equilibrium (QSE) for binding/unbinding reactions, we can analytically discern the impact of decoys on FPT statistics by using small noise approximation (SNA) and reformulating the FPT question in terms of a suitable variable whose dynamics is linear. The stability of the decoy-bound TFs influences the impact of decoys on FPT statistics. The presence of decoys makes the mean FPT long and even longer for unstable bound TFs. Decoys reduce noise in FPT, and stable decoy-bound TFs offer greater timing precision with less expression cost than their unstable counterparts. Interestingly, when both bound and free TFs decay at the same rate, the noise in FPT is not directly influenced by the number of decoys or their binding affinities. We verify these results by performing exact stochastic simulations. These results have important implications for the precise temporal scheduling of events involved in the functioning of biomolecular clocks, development processes, cell-cycle control, and cell-size homeostasis.

systems biology↗

bioRxiv2024

Segatella copri strains adopt distinct roles within a single individual's gut

Segatella copri is a dominant member of individuals gut microbiomes worldwide, especially in non-Western populations. Although metagenomic assembly and genome isolation have shed light on the genetic diversity of S. copri, the lack of available isolates from this clade has resulted in a limited understanding of how members genetic diversity translates into phenotypic diversity. Within the confines of a single gut microbiome, we have isolated 63 strains from diverse lineages of S. copri. We performed comparative analyses that exposed differences in cellular morphologies, preferences in polysaccharide utilization, yield of short-chain fatty acids, and antibiotic resistance across isolates. We further show that exposure to S. copri lineages either evokes strong or muted transcriptional responses in human intestinal epithelial cells. Our study exposes large phenotypic differences within related S. copri isolates, extending this to host-microbe interactions.

microbiology↗

bioRxiv2024

Phytoplasma Targeting of MADS-Box Factor SVP Suppresses Leaf Responses to Insect Vector Males, Promoting Female Attraction and Colonization

Obligate parasites often trigger significant changes in their hosts to facilitate transmission to new hosts. The molecular mechanisms behind these extended phenotypes - where genetic information of one organism is manifested as traits in another - remain largely unclear. This study explores the role of the virulence protein SAP54, produced by parasitic phytoplasmas, in attracting leafhopper vectors. SAP54 is responsible for the induction of leaf-like flowers in phytoplasma-infected plants. However, we previously demonstrated that the insects were attracted to leaves and the leaf-like flowers were not required. Here we made the surprising discovery that leaf exposure to leafhopper males is required for the attraction phenotype, suggesting a leaf response that distinguishes leafhopper sex in the presence of SAP54. In contrast, this phytoplasma effector alongside leafhopper females discourages further female colonization. We demonstrate that SAP54 effectively suppresses biotic stress response pathways in leaves exposed to the males. Critically, the host plant MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) emerges as a key element in the female leafhopper preference for plants exposed to males, with SAP54 promoting the degradation of SVP. This preference extends to female colonization of male-exposed svp null mutant plants over those not exposed to males. Our research underscores the dual role of the phytoplasma effector SAP54 in host development alteration and vector attraction - integral to the phytoplasma life cycle. Importantly, we clarify how SAP54, by targeting SVP, heightens leaf vulnerability to leafhopper males, thus facilitating female attraction and subsequent plant colonization by the insects. SAP54 essentially acts as a molecular "matchmaker," helping male leafhoppers more easily locate mates by degrading SVP-containing complexes in leaves. This study not only provides insights into the long reach of single parasite genes in extended phenotypes, but also opens avenues for understanding how transcription factors that regulate plant developmental processes intersect with and influence plant-insect interactions.

plant biology↗

bioRxiv2024

Impedance-based assay for pan-cancer early and rapid detection of cell-free DNA

Aberrant DNA methylation is a hallmark of cancer, and plasma cell-free DNA (cfDNA) containing these abnormal methylation patterns has emerged as a promising non-invasive biomarker for pan-cancer detection. However, intrinsic challenges remain that continue to limit its broad clinical application. Here we show a simple and rapid impedance biosensor called Asima Rev that can detect cancer cfDNA in under 5 min without the need for any molecular labelling, electrode modification, signal amplification, or target enrichment steps. Using 216 clinical samples (50 healthy) from 15 different cancer types (all stages) we show an overall sensitivity and specificity of 96.4% and 94.0%, respectively. Differences in methylation content between cancerous and healthy cfDNA lead to distinct solvation behaviour and electro-physicochemical property that remain consistent across cancer types regardless of the distribution patterns of methyl cytosine. Our test exploits this inherent difference.

biophysics↗

bioRxiv2024

Chloroquine Up-regulates Expression of SARS-CoV-2 receptor Angiotensin Converting Enzyme-2 in Endothelial Cells

Background and PurposeThe novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) posed a serious threat to global public health. Hydroxychloroquine (HCQ), which is a derivative of Chloroquine (CQ), was a WHO-recommended drug to treat COVID-19 with mixed effects. The purpose of the present study is to evaluate the plausible mechanisms of HCQ actions behind its observed mixed effect. Key ResultsWe demonstrate that CQ-treatment significantly up-regulates mesenchymal markers and SARS-CoV-2 receptor ACE2 in cultured endothelial cells. Conclusions & ImplicationsThe detrimental effect of HCQ in seriously ill COVID-19 patients might be due to CQ-induced increased expression of endothelial ACE2 exacerbating the severity of SARS-CoV-2 infection. Our study warrants further investigation in animal models and humans and caution while prescribing HCQ to patients with an impaired renin-angiotensin-aldosterone system, such as in hypertension, cardiovascular diseases, or chronic kidney disease; particularly with ACE-inhibitors or statin therapy.

cell biology↗

bioRxiv2024

Brain rewiring during developmental transitions: A Comparative Analysis of Larva and Adult Drosophila melanogaster

The brains ability to adapt through structural rewiring during developmental transitions is a fundamental aspect of neuroscience. Our study conducts a detailed comparison of Drosophila melanogaster s brain networks during larval and adult stages, revealing significant changes in neuronal wiring during developmental phases. The degree distribution of the larval brain deviates significantly from power-law behavior and fits well with the Weibull distribution. In contrast, the adult brain exhibits power-law behavior in its degree distribution, with the exponent for the out-degree distribution lying in the scale-free regime and the exponent for the in-degree distribution being close to this regime. This difference reflects a change in the robustness of brain development from larval to adult phases. The core of these networks also changes during development in terms of their cell composition and topological influence. The larval core comprises Mushroom Body neurons, while the adult core mainly has Antennal Lobe neurons. Moreover, all the core neurons in the larval brain are also part of the rich-club neurons, a group of neurons with high in/out degrees that are well connected, whereas the same is not true for the adult brain network. Additionally, the core of the larval brain displays a more heterogeneous connectivity profile in its second-order neighbors compared to adult brain neurons, indicating greater diversity in larval brain connectivity. Our work stands as a step forward in understanding the rewiring of brain networks across the life stages of Drosophila melanogaster.

neuroscience↗

bioRxiv2024

Spatial Transcriptomics in Mechanomics: New Horizons in Exploring the Mechanoregulation of Bone Regeneration

In recent decades, the field of bone mechanobiology has sought experimental techniques to unravel the molecular mechanisms governing the phenomenon of mechanically-regulated fracture healing. Each cell within a fracture site resides within different local micro-environments characterized by different levels of mechanical strain - thus, preserving the spatial location of each cell is critical in relating cellular responses to mechanical stimuli. Our spatial transcriptomics based "mechanomics" platform facilitates spatially-resolved analysis of the molecular profiles of cells with respect to their local in vivo mechanical environment by integrating time-lapsed in vivo micro-computed tomography, spatial transcriptomics, and micro-finite element analysis. We investigate the transcriptomic responses of cells as a function of the local strain magnitude by identifying the differential expression of genes in regions of high and low strain within a fracture site. Our platform thus has the potential to address fundamental open questions within the field and to discover mechano-responsive targets to enhance fracture healing.

physiology↗

bioRxiv2024

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics

Ebola virus (EBOV) is a high-consequence filovirus that gives rise to frequent epidemics with high case fatality rates and few therapeutic options. Here, we applied image-based screening of a genome-wide CRISPR library to systematically identify host cell regulators of Ebola virus infection in 39,085,093 million single cells. Measuring viral RNA and protein levels together with their localization in cells identified over 998 related host factors and provided detailed information about the role of each gene across the virus replication cycle. We trained a deep learning model on single-cell images to associate each host factor with predicted replication steps, and confirmed the predicted relationship for select host factors. Among the findings, we showed that the mitochondrial complex III subunit UQCRB is a post-entry regulator of Ebola virus RNA replication, and demonstrated that UQCRB inhibition with a small molecule reduced overall Ebola virus infection with an IC50 of 5 M. Using a random forest model, we also identified perturbations that reduced infection by disrupting the equilibrium between viral RNA and protein. One such protein, STRAP, is a spliceosome-associated factor that was found to be closely associated with VP35, a viral protein required for RNA processing. Loss of STRAP expression resulted in a reduction in full-length viral genome production and subsequent production of non-infectious virus particles. Overall, the data produced in this genome-wide high-content single-cell screen and secondary screens in additional cell lines and related filoviruses (MARV and SUDV) revealed new insights about the role of host factors in virus replication and potential new targets for therapeutic intervention.

microbiology↗