Biology subjects

Roy, S.

Publications and source records attributed to Roy, S..

At least 217 records · Page 12Linked to original sources

bioRxiv2022

Inference of cell type-specific gene regulatory networks on cell lineages from single cell omic datasets

Cell type-specific gene expression patterns are outputs of transcriptional gene regulatory networks (GRNs) that connect transcription factors and signaling proteins to target genes. Single-cell technologies such as single cell RNA-sequencing (scRNA-seq) and single cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq), can examine cell-type specific gene regulation at unprecedented detail. However, current approaches to infer cell type-specific GRNs are limited in their ability to integrate scRNA-seq and scATAC-seq measurements and to model network dynamics on a cell lineage. To address this challenge, we have developed single-cell Multi-Task Network Inference (scMTNI), a multi-task learning framework to infer the GRN for each cell type on a lineage from scRNA-seq and scATAC-seq data. Using simulated and real datasets, we show that scMTNI is a broadly applicable framework for linear and branching lineages that accurately infers GRN dynamics and identifies key regulators of fate transitions for diverse processes such as cellular reprogramming and differentiation.

bioinformatics↗

bioRxiv2022

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo. SIGNIFICANCE STATEMENTModifying the DNA of cells in the brain could present opportunities for new treatments of neurological diseases. In this report, we describe a nanocapsule system designed to deliver the elements needed to modify the DNA of brain cells, also known as genome editing. These nanocapsules are created by chemically encapsulating the genome editing components, such that the nanocapsules are stable when prepared and biodegradable to release their payload upon entering cells. When injected into the mouse brain, our research shows that the nanocapsules lead to safe and efficient editing of DNA in neurons.

neuroscience↗

bioRxiv2022

Imaging cortical engagement during motor imagery, mental arithmetic, and silent word generation using MEG beta rhythm

Accurate mapping of cortical engagement during mental imagery or cognitive tasks remains a challenging brain-imaging problem with immediate relevance to the development of brain-computer interfaces (BCI). We analyzed data from fourteen individuals who performed cued motor imagery, mental arithmetic, or silent word generation tasks during MEG recordings. During the motor imagery task, participants imagined the movement of either both hands (HANDS) or both feet (FEET) after the appearance of a static visual cue. During the cognitive task, participants either mentally subtracted two numbers that were displayed on the screen (SUB) or generated words starting with a letter cue that was presented (WORD). The MEG recordings were denoised using a combination of spatiotemporal filtering, the elimination of noisy epochs, and ICA decomposition. Cortical source power in the beta-band (17-25 Hz) was estimated from the selected temporal windows using a frequency-resolved beamforming method applied to the sensor-level MEG signals. The task-related cortical engagement was inferred from beta power decrements within non-overlapping 400 ms temporal windows between 400 and 2800 ms after cue presentation relative to a baseline 400 ms temporal window before cue onset. We estimated the significance of these power changes within each parcel of the Desikan-Killiany atlas using a non-parametric permutation test at the group level. During the HANDS and FEET movement-imagery conditions, beta-power decreased in premotor and motor areas, consistent with a robust engagement of these cortical regions during motor imagery. During WORD and SUB tasks, beta-power decrements signaling cortical engagement were lateralized to left hemispheric brain areas that are expected to engage in language and arithmetic processing within the temporal (superior temporal gyrus), parietal (supramarginal gyrus), and (inferior) frontal regions. A leave-one-subject-out cross-validation using a support vector machine (SVM) applied to beta power decrements across brain parcels yielded accuracy rates of 74% and 68% respectively, for classifying motor-imagery (HANDS-vs-FEET) and cognitive (WORD-vs-SUB) tasks. From the motor-versus-nonmotor contrasts, accuracy rates of 85% and 80% respectively, were observed for HAND-vs-WORD and HAND-vs-SUB. A multivariate Gaussian process classifier (GPC) provided an accuracy rate of 60% for a four-way (HANDS-FEET-WORD-SUB) classification problem. The regions identified by both SVM and GPC classification weight maps were largely consistent with the source modeling findings. Within-subject correlations of beta-decrements during the two task sessions provided insights into the level of engagement by individual subjects and showed moderately high correlations for most subjects. Our results show that it is possible to map the dynamics of cortical engagement during mental processes in the absence of dynamic sensory stimuli or overt behavioral outputs using task-related beta-power decrements. The ability to do so with the high spatiotemporal resolution afforded by MEG could potentially help better characterize the physiological basis of motor or cognitive impairments in neurological disorders and guide strategies for neurorehabilitation.

neuroscience↗

bioRxiv2022

The effect of environment on the evolution and proliferation of protocells of increasing complexity

The formation, growth, division and proliferation of protocells containing RNA strands is an important step in ensuring the viability of a mixed RNA-lipid world. Experiments and computer simulations indicate that RNA encapsulated inside protocells can favour the protocell promoting its growth while protecting the system from being over-run by parasites. Recent work has also shown the rolling-circle replication mechanism can be harnessed to ensure rapid growth of RNA strands and probabilistic emergence and proliferation of protocells with functionally diverse ribozymes. Despite these advances in our understanding of a primordial RNA-lipid world, key questions remain about the ideal environment for formation of protocells and its role in regulating the proliferation of functionally complex protocells. The hot spring hypothesis suggests that mineral-rich regions near hot-springs, subject to dry-wet cycles provide an ideal environment for the origin of primitive protocells. We develop a computational model to study protocellular evolution in such environments that are distinguished by the occurrence of three distinct phases, a wet phase, followed by a gel phase, and subsequently by a dry phase. We determine the conditions under which protocells containing multiple types of ribozymes can evolve and proliferate in such regions. We find that diffusion in the gel-phase can inhibit the proliferation of complex protocells with the extent of inhibition being most significant when a small fraction of protocells is eliminated during environmental cycling. Our work clarifies how the environment can shape the evolution and proliferation of complex protocells.

evolutionary biology↗

bioRxiv2022

Rapid non-destructive method to phenotype stomatal traits

BackgroundStomata are tiny pores on the leaf surface that are central to gas exchange. Stomatal number, size and aperture are key determinants of plant transpiration and photosynthesis, and variation in these traits can affect plant growth and productivity. Current methods to screen for stomatal phenotypes are tedious and not high throughput. This impedes research on stomatal biology and hinders efforts to develop resilient crops with optimised stomatal patterning. We have developed a rapid non-destructive method to phenotype stomatal traits in four species: wheat, rice, tomato and Arabidopsis. ResultsThe method consists of two steps. The first is the non-destructive capture of images of the leaf surface from plants in their growing environment using a handheld microscope; a process which only takes a few seconds compared to minutes for other methods. The second is to analyse stomatal features using a machine learning model that automatically detects, counts and measures stomatal number, size and aperture. The accuracy of the machine learning model in detecting stomata ranged from 76% to 99%, depending on the species, with a high correlation between measures of number, size and aperture between measurements using the machine learning models and by measuring them manually. The rapid method was applied to quickly identify contrasting stomatal phenotypes. ConclusionsWe developed a method that combines rapid non-destructive imaging of leaf surfaces with automated image analysis. The method provides accurate data on stomatal features while significantly reducing time for data acquisition and analysis. It can be readily used to phenotype stomata in large populations in the field and in controlled environments.

plant biology↗

bioRxiv2022

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is stable among repeated serial samples from the same host, is transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.

evolutionary biology↗

bioRxiv2022

Assessing combinatorial diversity of aureochrome bZIPs through genome-wide screening

Aureochromes are unique blue light-responsive LOV (Light Oxygen Voltage) photoreceptors cum basic leucine zipper (bZIP) transcription factors (TFs), present exclusively in photosynthetic marine stramenopiles. Considering the availability of the complete genome sequence, this study focuses particularly on aureochromes from Ectocaupus siliculosus. Aureochromes mediate light-regulated developmental responses in this brown photosynthetic algae. Both the LOV sensor and the bZIP effector shows sequence-structure conservation. The LOV+bZIP modules of aureochrome homologs/ paralogs are not only structurally similar but also show an identical oligomeric state -- preferably dimeric. Aureochromes execute diverse cellular responses in different photosynthetic stramenopiles-- though their activities can vary even within a given algal species. Besides a heterogeneous linker connecting the sensor-effector and a flexible N-terminal region, the sequence composition of both the domains is vital. Therefore, it is important to understand whether aureochromes select dimerization partners from the same family or interact with other bZIPs as well. To regulate multifarious bio-logical activities, it is possible that aureochromes activate the global TF interaction network. Following homo/heterodimer modeling, we address the compatibility of dimerization partners by screening through heptad repeats. We evaluate the dimer interface area in terms of gain in solvation energy as well as the number of hydrogen bonds/salt bridge interactions. We further explore the relative stability of these structures from a graph-theoretic perspective through well-studied measures such as the energy of the graph and average participation coefficient. Furthermore, we also conduct an information-theoretic analysis using network information centrality and Kullback-Leibler divergence. We find that all our investigations into the relative stability of these dimers using diverse methods from bioinformatics, network science, and, information theory are in harmonious agreement. Our approach and findings should facilitate the design of experiments.

bioinformatics↗

bioRxiv2022

HIV positive patients on antiretroviral therapy have an altered mucosal intestinal but not oral microbiome

This study characterized compositional and functional shifts in the intestinal and oral microbiome in HIV-positive patients compared to uninfected individuals. 79 specimens were collected from 5 HIV-positive and 12 control subjects from five locations (colon brush, colon wash, TI brush, TI wash and saliva) during colonoscopy and at patient visits. Microbiome composition was characterized using 16S rRNA sequencing and microbiome function was predicted using bioinformatics tools (PICRUSt and Bugbase). Our analysis indicated that the {beta} diversity of all intestinal samples (colon brush, colon wash, TI brush, TI wash) from patients with HIV was significantly different from patients without HIV, as measured by weighted UniFrac distances. Specifically, bacteria from genera Prevotella, Fusobacterium, Eubacterium, Megasphaera, Mogibacterium and Mitsuokella were more abundant in samples from HIV-positive patients. On the other hand, bacteria from genera Ruminococus, Blautia, and Clostridium were more abundant in samples from HIV-negative patients. Additionally, HIV-positive patients had higher abundances of biofilm-forming and pathogenic bacteria. Furthermore, pathways related to translation and nucleotide metabolism were elevated in HIV-positive patients whereas pathways related to lipid and carbohydrates metabolism and membrane transport were positively correlated with samples from HIV-negative patients. Our analyses further showed variations in microbiome composition in HIV-positive and negative patients by sampling site, with samples from colon wash, colon brush and TI wash significant between groups while samples from TI brush and saliva were not significant. Taken together, here we report altered intestinal microbiome composition and function in patients with HIV compared to uninfected patients, though we found no changes in the oral microbiome. Author summaryOver 37 million people worldwide are living with HIV. Although the availability of antiretroviral therapy has significantly reduced the number of AIDS-related deaths, individuals living with HIV are at increased risk for opportunistic infections. We now know that HIV interacts with the trillions of bacteria, fungus, and viruses in the human body termed the microbiome. The advent of next generation sequencing has allowed us to characterize the composition of the microbiome and how HIV changes the microbiome composition to influence disease severity and progression. Previous studies have examined changes in the microbiome in HIV-positive and negative individuals. However, only a limited number of studies have compared variations in the oral and gastrointestinal microbiome with HIV infection. Furthermore, very few studies have looked at how the microbiome in HIV infection may vary by sampling site. Here, we detail how the oral and gastrointestinal microbiome changes with HIV infection and use 5 different sampling sites to gain a more comprehensive view of these changes by location. Our results show site-specific changes in the intestinal microbiome associated with HIV infection. Additionally, we show that while there are significant changes in the intestinal microbiome, there are no significant changes in the oral microbiome.

microbiology↗

bioRxiv2022

The peptide GOLVEN10 controls nodule and lateral root organogenesis and positioning along the longitudinal root axis

O_LIGLV/RGF peptide encoding genes can be identified in genomes of all plants that can form roots or root-like structures suggesting they were essential for transition of plants to land. C_LIO_LIIn Medicago truncatula, five of fifteen GOLVEN(GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide coding genes were induced during nodule organogenesis and to a varying extent under nitrogen deficiency and auxin treatment. Expression of MtGLV9 and MtGLV10 at nodule initiation sites was dependent on the transcription factor NODULE INCEPTION. C_LIO_LIOverexpression of all five nodule-induced GLV genes in M. truncatula hairy roots as well as application of the corresponding synthetic peptides resulted in a 25-50% reduction in nodule number indicating GOLVENs are negative regulators of nodule organogenesis. C_LIO_LIThe peptide GOLVEN10 shifted the position of the first formed lateral root (rhizotaxis) as well as the first formed nodule along the longitudinal primary root axis, a phenomenon we term nodulotaxis, thereby reducing the absolute length of the zone of lateral organ formation on roots. C_LIO_LIApplication of synthetic GOLVEN10 peptide caused an increase in cell number but not cell length in each root cortical cell layer causing an increase in root length and a consequent spatiotemporal delay in formation of the first lateral organ. C_LI Plain Language SummaryNodule positioning is an understudied trait, yet it determines the length of the root that can support nodule formation and consequently the total number of functional nodules formed. We identify for the first time, genetic factors called GOLVEN peptides that alter nodule and lateral root positioning on the primary root along with several other traits including nodule organ initiation and root architecture.

plant biology↗

bioRxiv2022

TCGADEPMAP -- Mapping Translational Dependencies and Synthetic Lethalities within The Cancer Genome Atlas

The Cancer Genome Atlas (TCGA) has yielded unprecedented genetic and molecular characterization of the cancer genome, yet the functional consequences and patient-relevance of many putative cancer drivers remain undefined. TCGADEPMAP is the first hybrid map of translational tumor dependencies that was built from machine learning of gene essentiality in the Cancer Dependency Map (DEPMAP) and then translated to TCGA patients. TCGADEPMAP captured well-known and novel cancer lineage dependencies, oncogenes, and synthetic lethalities, demonstrating the robustness of TCGADEPMAP as a translational dependency map. Exploratory analyses of TCGADEPMAP also unveiled novel synthetic lethalities, including the dependency of PAPSS1 driven by loss of PAPSS2 which is collaterally deleted with the tumor suppressor gene PTEN. Synthetic lethality of PAPSS1/2 was validated in vitro and in vivo, including the underlying mechanism of synthetic lethality caused by the loss of protein sulfonation that requires PAPSS1 or PAPSS2. Moreover, TCGADEPMAP demonstrated that patients with predicted PAPSS1/2 synthetic lethality have worse overall survival, suggesting that these patients are in greater need of drug discovery efforts to target PAPSS1. Other map "extensions" were built to capture unique aspects of patient-relevant tumor dependencies using the flexible analytical framework of TCGADEPMAP, including translating gene essentiality to drug response in patient-derived xenograft (PDX) models (i.e., PDXEDEPMAP) and predicting gene tolerability within normal tissues (GTEXDEPMAP). Collectively, this study demonstrates how translational dependency maps can be used to leverage the rapidly expanding catalog of patient genomic datasets to identify and prioritize novel therapeutic targets with the best therapeutic indices.

cancer biology↗

bioRxiv2022

Nematode histone H2A variant evolution reveals diverse histories of retention and loss, and evidence for conserved core-like variants

Histone variants are paralogs that replace canonical histones in nucleosomes, often imparting novel functions. Despite their importance, how histone variants arise and evolve is poorly understood. Reconstruction of histone protein evolution is challenging due to high amino acid conservation and large differences in evolutionary rates across gene lineages and sites. Here we combined amino acid sequences and intron position data from 108 nematode genomes to trace the evolutionary histories of the three H2A variants found in Caenorhabditis elegans: the ancient H2A.ZHTZ-1, the sperm-specific HTAS-1, and HIS-35, which differs from canonical H2A by a single glycine-to-alanine C-terminal change. We find disparate evolutionary histories. Although the H2A.ZHTZ-1 protein is highly conserved, its gene exhibits recurrent intron gain and loss. This pattern suggests that it is intron presence, rather than specific intron sequences or positions, that may be important to H2A.Z functionality. In contrast, for HTAS-1 and HIS-35, we find variant-specific intron positions that are conserved across species. HIS-35 arose in the ancestor of Caenorhabditis and its sister group, including the genus Diploscapter, while the sperm-specific variant HTAS-1 arose more recently in the ancestor of a subset of Caenorhabditis species. HIS-35 exhibits gene retention in some descendent lineages but also recurrent gene loss in others, suggesting that histone variant use or functionality is highly flexible in this case. We also find that the single amino acid differentiating HIS-35 from core H2A is ancestral and common across canonical Caenorhabditis H2A sequences and identify one nematode species that bear identical HIS-35 and canonical H2A proteins, findings that are not predicted from the hypothesis that HIS-35 has a distinct function. Instead, we speculate that HIS-35 enables H2A expression across the cell cycle or in distinct tissues; genes encoding such partially-redundant functions may be advantageous yet relatively replaceable over evolutionary times, consistent with the patchwork pattern of retention and loss of both genes. Our study shows the evolutionary trajectory for histone H2A variants with distinct functions and the utility of intron positions for reconstructing the evolutionary history of gene families, particularly those undergoing idiosyncratic sequence evolution.

molecular biology↗

bioRxiv2022

Functional substitutability of native herbivores by livestock for long-term soil carbon sequestration depends on microbial decomposers.

Grazing by large mammalian herbivores impacts climate as it can favor the size and stability of a large carbon (C) pool in soil. As native herbivores in the worlds grasslands, steppes, and savannas are progressively being displaced by livestock, it is important to ask whether livestock can emulate the functional roles of their native counterparts. While livestock and native herbivores can have remarkable similarity in their traits, they can differ greatly in their impacts on vegetation composition which can affect soil-C. It is uncertain how their similarities and differences impact soil-C via their influence on microbial decomposers. We test competing alternative hypotheses with a replicated, long-term, landscape-level, grazing-exclusion experiment to ask whether livestock in the Trans-Himalayan ecosystem of northern India can match decadal-scale (2005-2016) soil-C stocks under native herbivores. We evaluate multiple lines of evidence from 17 variables that influence soil-C (quantity and quality of C-input from plants, microbial biomass and metabolism, microbial community composition, veterinary antibiotics in soil), and asses their inter-relationships. Livestock and native herbivores differed in their effects on several soil microbial processes. Microbial carbon use efficiency (CUE) was 19% lower in soils under livestock. Compared to native herbivores, areas used by livestock contained 1.5 kg C m-2 less soil-C. Structural equation models showed that alongside effects arising from plants, livestock alter soil microbial communities which is detrimental for CUE, and ultimately also for soil-C. Supporting evidence pointed toward a link between veterinary antibiotics used on livestock, microbial communities, and soil-C. Overcoming the challenges of sequestrating antibiotics to minimize their potential impacts on climate, alongside microbial rewilding under livestock, may reconcile the conflicting demands from food-security and ecosystem services. Conservation of native herbivores and better management of livestock is crucial for soil-C stewardship to envision and achieve natural climate solutions.

ecology↗

bioRxiv2022

Preclinical stage abundance and nuclear antigen reactivity of fecal Immunoglobulin A (IgA) varies among males and females of lupus-prone mouse models

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies with nuclear antigen (nAg) specificity. Using (SWRxNZB)F1 (SNF1) mice, we showed higher levels of IgA production in the intestine and the nAg reactivity of fecal IgA under lupus susceptibility. Here, we determined if the fecal IgA abundance and nAg reactivity are higher in, different among, various lupus-prone preclinical models (MRL/lpr, NZBxNZW-F1, SNF1, NZM2410 and NZM2328). We also determined if the fecal IgA nAg reactivity at pre-seropositive ages correlates with the eventual serum autoantibody levels in males and females of these mouse models. We show that age dependent increase in the abundance and nAg reactivity of fecal IgA can vary among different lupus-prone mouse models. Importantly, fecal IgA in these mice show significant levels of nAg reactivity, starting as early as at juvenile age. Furthermore, the pre-seropositive stage nAg reactivity of fecal IgA in most lupus-prone strains correlates well with that of eventual, seropositive stage systemic autoantibody levels. Gender differences in serum autoantibody levels were preceded by similar differences in the fecal IgA abundance and nAg reactivity. These observations suggest that fecal IgA features, nAg reactivity particularly, could serve as a biomarker for early prediction of the eventual systemic autoimmunity in lupus-prone subjects.

immunology↗

bioRxiv2022

Nutrient and salt depletion synergistically boosts glucose metabolism in individual Escherichia coli cells

The interaction between a cell and its environment shapes fundamental intracellular processes such as cellular metabolism. In most cases growth rate is treated as a proximal metric for understanding the cellular metabolic status. However, changes in growth rate might not reflect metabolic variations in individuals responding to environmental fluctuations. Here we use single-cell microfluidics-microscopy combined with transcriptomics, proteomics and mathematical modelling to quantify the accumulation of glucose within Escherichia coli cells. In contrast to the current consensus, we reveal that environmental conditions which are comparatively unfavourable for growth, where both nutrients and salinity are depleted, increase glucose accumulation rates in individual bacteria and population subsets. We find that these changes in metabolic function are underpinned by variations at the translational and posttranslational level but not at the transcriptional level and are not dictated by changes in cell size. The metabolic response-characteristics identified greatly advance our fundamental understanding of the interactions between bacteria and their environment and have important ramifications when investigating cellular processes where salinity plays an important role.

microbiology↗

bioRxiv2022

Prostaglandin E1 as therapeutic molecule for Nephronophthisis and related ciliopathies

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using an in cellulo medium-throughput drug-screen, we identified 51 FDA-approved compounds and selected 11 for their physicochemical properties, including prostaglandin E1 (PGE1). PGE1 was further validated to rescue ciliogenesis in immortalized patient NPHP1-/- urine-derived renal tubular cells and corroborated by the effects of its analog PGE2. The two molecules reduced pronephric cyst occurrence in vivo in nphp4 zebrafish model, and PGE1 treatment in Nphp1-/- mice led to a significant reduction of renal tubular dilatations, partially restoring cilia length within tubules. Finally, comparative transcriptomics allowed identification of key molecules downstream PGE1. Altogether, our drug-screen strategy led to the identification of PGE1 as the first potential therapeutic molecule for NPH-associated ciliopathies. Significant statementJuvenile nephronophthisis (NPH) is a renal ciliopathy due to a dysfunction of primary cilia and a common genetic cause of end-stage renal disease in children and young adults. No curative treatment is available. This paper describes the identification of Prostaglandin E1 (PGE1) as the first potential therapeutic molecule for NPH-associated ciliopathies. We demonstrated that PGE1 rescues defective ciliogenesis and ciliary composition in NPHP1-/- patient urine-derived renal tubular cells. Furthermore, PGE1 improves ciliary and kidney phenotypes in our NPH zebrafish and Nphp1-/- mouse models. Finally, in vitro experiments as well as transcriptomic analyses pointed out several pathways downstream PGE1 as cAMP, cell-cell/cell-matrix adhesion or actin cytoskeleton. Altogether, our findings provide a new alternative for treatment of NPH.

cell biology↗

bioRxiv2022

Discovery of novel compounds as potent activators of Sirt3

Among the sirtuin enzymes, Sirt3 is one of the most important deacetylases as it regulates acetylation levels in mitochondria, which are linked to the metabolism of multiple organs and therefore involved in many types of age-related human diseases such as cancer, heart diseases and metabolic diseases. Given the dearth of direct activators of Sirt3, the identification of new modulators could be a key step in the development of new therapeutics. Here we report the discovery of Sirt3 modulators, including activators, through the use of DNA encoded library technology (DEL). The most enriched compounds after DEL selection against SIRT3 were evaluated according to their activity and affinity. Our best activator seems at least as potent as Honokiol (HKL) while the docking studies suggest that our modulators interact with Sirt3 at an atypical site. Our results establish the attractiveness of the DEL technology in identifying novel and potent Sirt3 activators and, therefore, in associated therapeutic applications.

biochemistry↗

bioRxiv2022

Development, Structure, and Mechanism of Synthetic Antibodies that Target Claudin and Clostridium perfringens Enterotoxin Complexes

Strains of the Gram-positive bacterium Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflict millions of humans and domesticated animals annually by causing prevalent gastrointestinal illnesses. CpEs C-terminal domain (cCpE) binds cell surface receptors then its N-terminal domain restructures to form a membrane-penetrating {beta}-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are the receptors for CpE, and also control the architecture and function of cell/cell contacts called tight junctions that create barriers to intercellular transport of solutes. CpE binding disables claudin and tight junction assembly and induces cytotoxicity via {beta}-pore formation, disrupting gut homeostasis. Here, we aimed to develop probes of claudin/CpE assembly using a phage display library encoding synthetic antigen-binding fragments (sFabs) and discovered two that bound complexes between human claudin-4 and cCpE. We established each sFabs unique modes of molecular recognition, their binding affinities and kinetics, and determined structures for each sFab bound to ~35 kDa claudin-4/cCpE in three-protein comprised complexes using cryogenic electron microscopy (cryoEM). The structures reveal a recognition epitope common to both sFabs but also that each sFab distinctly conforms to bind their antigen, which explain their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in further binding changes. Together, these findings validate the structures and uncover the mechanism of targeting claudin-4/cCpE complexes by these sFabs. Based on these structural insights we generate a model for CpEs cytotoxic claudin-bound {beta}-pore that predicted that these two sFabs would not prevent CpE cytotoxicity, which we verify in vivo with a cell-based assay. This work demonstrates the development and targeting mechanisms of sFabs against claudin/cCpE that enable rapid structural elucidation of these small membrane protein complexes using a cryoEM workflow. It further provides a structure-based framework and therapeutic strategies for utilizing these sFabs as molecular templates to target claudin/CpE assemblies, obstruct CpE cytotoxicity, and treat CpE-linked gastrointestinal diseases that cause substantial economic and quality of life losses throughout the world.

biophysics↗

bioRxiv2021

Capturing a crucial 'disorder-to-order transition' at the heart of the coronavirus molecular pathology - triggered by highly persistent, interchangeable salt-bridges

The COVID-19 origin debate has greatly been influenced by Genome comparison studies of late, revealing the seemingly sudden emergence of the Furin-Like Cleavage Site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCSSpike) containing its 681PRRAR685 motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a disorder-to-order transition of the FLCSSpike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike-Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCSSpike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seems the other key characteristic features of the Spike-Furin binding in SARS-CoV-2 allowing the system to breathe - a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike-Furin binding events generally in the coronavirus lineage seems to have a preference for ionic bond formation, even with lesser number of cationic residues at their potentially polybasic FLCSSpike patches. The interaction energies are suggestive of a characteristic metastabilities attributed to Spike-Furin interactions generally to the coronavirus lineage - which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. T he current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution but also add substantially to the existing theories of proteolytic cleavages.

biophysics↗