Biology subjects

Patel, A.

Publications and source records attributed to Patel, A..

At least 127 records · Page 7Linked to original sources

bioRxiv2021

Tumor protein D54 binds intracellular nanovesicles via an amphipathic lipid packing sensor (ALPS) motif

Tumor Protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54 and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical transport vesicles (e.g. COPI and COPII). Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by several amphipathic helices, which could fold upon binding to lipid membranes. One of these helices has the physicochemical features of an Amphipathic Lipid Packing Sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Limited proteolysis, CD spectroscopy, tryptophan fluorescence and cysteine mutagenesis coupled to covalent binding of a membrane sensitive probe show that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. TPD54 binding to artificial liposomes is very sensitive to liposome size and to lipid unsaturation but is poorly dependent on lipid charge. Cellular investigations confirmed the key role of the ALPS motif in vesicle targeting. Surprisingly, the vesicles selected by TPD54 poorly overlap with those captured by the golgin GMAP-210, a long vesicle tether at the Golgi apparatus, which displays a dimeric coiled-coil architecture and an N-terminal ALPS motif. We propose that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and -independent mechanisms.

cell biology↗

bioRxiv2021

Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.

immunology↗

bioRxiv2021

Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche

Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Discrete AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.

developmental biology↗

bioRxiv2021

CRISPR-Cas9 mediated knockout of SagD gene for overexpression of streptokinase in Streptococcus equisimilis

Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (Heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is high globally than the production due to increased incidences of various heart conditions. The main source of streptokinase is from various strains of Streptococcus. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD inhibitor gene for production of streptokinase that needs to be knocked out in order to increase it expression. However, FasX is a small RNA (sRNA) present in group A Streptococcus species which is responsible for post-transcriptional regulation of streptokinase (ska) gene by binding at the 5 end of ska mRNA. S. equisimilis is a {beta}-hemolysin producing streptococcus bacterium (group C) containing the orthologue of FasX and natively expresses a clinically important thrombolytic streptokinase. In order to improve the stability of mRNA and increasing the expression of streptokinase which is inhibited by SagD. We used CRISPR-Cas9 to successfully knock-out of SagD gene and observed a 13.58-fold relative quantification of streptokinase expression in the mutant strain as compared to wild type. We have also demonstrated the successful target gene knockout using CRISPR-Cas9 in S. equisimilis that engineered strain can be used further for overexpression of streptokinase for therapeutic applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/465095v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@8e3ebeorg.highwire.dtl.DTLVardef@b92f4eorg.highwire.dtl.DTLVardef@1d0dd8borg.highwire.dtl.DTLVardef@180ee54_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Spatial and temporal analysis of neutrophil trans-epithelial migration in response to RSV infection

The recruitment of neutrophils to the infected airway occurs early following respiratory syncytial virus (RSV) infection and high numbers of activated neutrophils in airway and blood is associated with the development of severe disease. Here, we investigated whether trans-epithelial migration across primary human airway epithelial cells (AECs) is sufficient and necessary for neutrophil activation during RSV infection. Using flow cytometry, we identified three populations of neutrophils in our in vitro model; those in suspension in basolateral and apical compartments and those that migrated and adhered to AECs. After 1h incubation, the number of adherent neutrophils was significantly greater following RSV infection compared to mock infected. We found that, when migration occurred, neutrophil expression of CD11b, CD62L, CD64, NE and MPO increased in all compartments. However, this did not occur when neutrophils were prevented from migrating. This suggests that the heightened neutrophil activation we detected in the basolateral compartment may be due to reverse migrating neutrophils, as has been suggested by clinical observations. Using live-cell fluorescent microscopy, we then profiled the early temporal and spatial movement and adherence of human neutrophils during migration. Our findings suggest three main phases of early neutrophil recruitment and behaviour in the airways during RSV infection, with neutrophil recruitment, activation and adherence to RSV infected AECs, with clustering, occurring within the first 20 minutes. This work and the model we developed could provide new insight into how neutrophil activation and a dysregulated neutrophil response to RSV mediates disease severity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/463016v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16639c5org.highwire.dtl.DTLVardef@5aeeacorg.highwire.dtl.DTLVardef@1eb96ceorg.highwire.dtl.DTLVardef@1f9b899_HPS_FORMAT_FIGEXP M_FIG Building on previous work of neutrophil function we propose 3 main phases of early neutrophil recruitment and behaviour in the airways during RSV infection. Phase 1. Initial chemotaxis and adherence: Here unstimulated circulating neutrophils expressing baseline levels of CD11b migrate across infected AECs in response to chemotactic signals in the apical supernatant. Some neutrophils remain adherent to the infected AECs. Phase 2: Activation and reverse migration: once on the apical side of the epithelium, neutrophils increase expression of CD11b and other activation associated markers, and some activated neutrophils undergo reverse migration. Neutrophils with greater expression of CD11b are detected on the basolateral side Phase 3: Amplified chemotaxis and clustering: after 20 minutes, adherent neutrophils begin to rapidly cluster on RSV infected primary airway epithelial cells cultures, mediated by signalling from a dying neutrophil. Drawing created using BioRender.com. C_FIG

immunology↗

bioRxiv2021

Causal reasoning over knowledge graphs leveraging drug-perturbed and disease-specific transcriptomic signatures for drug discovery

Network-based approaches are becoming increasingly popular for drug discovery as they provide a systems-level overview of the mechanisms underlying disease pathophysiology. They have demonstrated significant early promise over other methods of biological data representation, such as in target discovery, side effect prediction and drug repurposing. In parallel, an explosion of -omics data for the deep characterization of biological systems routinely uncovers molecular signatures of disease for similar applications. Here, we present RPath, a novel algorithm that prioritizes drugs for a given disease by reasoning over causal paths in a knowledge graph (KG), guided by both drug-perturbed as well as disease-specific transcriptomic signatures. First, our approach identifies the causal paths that connect a drug to a particular disease. Next, it reasons over these paths to identify those that correlate with the transcriptional signatures observed in a drug-perturbation experiment, and anti-correlate to signatures observed in the disease of interest. The paths which match this signature profile are then proposed to represent the mechanism of action of the drug. We demonstrate how RPath consistently prioritizes clinically investigated drug-disease pairs on multiple datasets and KGs, achieving better performance over other similar methodologies. Furthermore, we present two applications showing how one can deconvolute the predictions made by RPath as well as predict novel targets. Finally, we have made the source code and data publicly available at https://github.com/enveda/RPath.

bioinformatics↗

bioRxiv2021

Profiling Transcription Initiation in Peripheral Leukocytes Reveals Severity-Associated Cis-Regulatory Elements in Critical COVID-19

The contribution of transcription factors (TFs) and gene regulatory programs in the immune response to COVID-19 and their relationship to disease outcome is not fully understood. Analysis of genome-wide changes in transcription at both promoter-proximal and distal cis-regulatory DNA elements, collectively termed the active cistrome, offers an unbiased assessment of TF activity identifying key pathways regulated in homeostasis or disease. Here, we profiled the active cistrome from peripheral leukocytes of critically ill COVID-19 patients to identify major regulatory programs and their dynamics during SARS-CoV-2 associated acute respiratory distress syndrome (ARDS). We identified TF motifs that track the severity of COVID- 19 lung injury, disease resolution, and outcome. We used unbiased clustering to reveal distinct cistrome subsets delineating the regulation of pathways, cell types, and the combinatorial activity of TFs. We found critical roles for regulatory networks driven by stimulus and lineage determining TFs, showing that STAT and E2F/MYB regulatory programs targeting myeloid cells are activated in patients with poor disease outcomes and associated with single nucleotide genetic variants implicated in COVID-19 susceptibility. Integration with single-cell RNA-seq found that STAT and E2F/MYB activation converged in specific neutrophils subset found in patients with severe disease. Collectively we demonstrate that cistrome analysis facilitates insight into disease mechanisms and provides an unbiased approach to evaluate global changes in transcription factor activity and stratify patient disease severity.

genomics↗

bioRxiv2021

Spontaneous emergence of non-convertible cell states with CD24-High phenotype results in phenotypic heterogeneity that associates with poor prognosis in oral cancer

PurposeCellular dynamics between phenotypically heterogeneous subpopulations of cancer cells within individual tumor is shown to be responsible for drug tolerance and overall poor prognosis; however, evidences were largely missing in oral cancer. Therefore, this study was undertaken to describe the dynamic phenotypic states among oral cancer cells, its influence on transcriptomic heterogeneity as well as its clinical significance. Experimental DesignWe multiplexed phenotypic markers of putative oral-stem-like cancer cells (SLCCs) and characterized diversity among CD44-positive oral cancer cell subpopulations with respect to distinct expression of CD24 and aldehyde dehydrogenase (ALDH)-activity in multiple cell lines. Population trajectories were characterized by Markov model and cell states were defined based on the population specific RNA sequencing (RNAseq). ssGSEA based gene expression signatures were explore for prognostic significance. ResultsOral cancer cells followed two distinct patterns of spontaneous repopulation dynamics with stochastic inter-conversions on ALDH-axis, however a strict non-interconvertible transition on CD24-axis. Interestingly, plastic ALDH-axis was harnessed to enrich ALDHHigh subpopulations in response to Cisplatin treatment, to adapt a drug tolerant state. Phenotype-specific RNAseq results suggested the organization of subpopulations into hierarchical structure with possible maintenance of intermediate states of stemness within the differentiating oral cancer cells. Further, survival analysis with each subpopulation-specific gene signature strongly suggested that the cell-state dynamics may act as possible mechanism to drive ITH, resulting in poor prognosis in patient. ConclusionsOur results emphasized the prognostic power of the population dynamics in oral cancer. Importantly, we have described the phenotypic-composition of heterogeneous subpopulations critical for global tumor behaviour in oral cancer; which is a prerequisite knowledge important for precision treatment, however largely lacking for most solid tumors. Graphical AbstractWe have characterized diversity among CD44-positive oral cancer cells lines with respect to distinct expression of CD24 and ALDH-activity. Subpopulations showed stochastic inter-conversions on ALDH-axis but a strict non-interconvertible transition of CD24Low to CD24High phenotype, even in response to chemotherapy-induced stress. RNAseq study suggested the organization of subpopulations into hierarchical structure with possible maintenance of intermediate alternate states of stemness within the differentiating oral cancer cells. The described population dynamics demonstrtaed influence tumor behaviour possibly by increasing intratumoral heterogeneity in aggressive oral tumors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/457509v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1dc6a7org.highwire.dtl.DTLVardef@dd0948org.highwire.dtl.DTLVardef@18c9603org.highwire.dtl.DTLVardef@cca8fb_HPS_FORMAT_FIGEXP M_FIG C_FIG Translational relevanceIntratumoral heterogeneity (ITH) has been the clinically important factor, impacting aggressive cancer behaviour, drug tolerance and overall poor prognosis. Recent high-throughput studies have provided better cellular and molecular resolution of ITH; however, the prerequisite knowledge which defines the composition of subpopulations critical for global tumor behaviour is majorly lacking for most of the solid tumors. By combining phenotypic markers, we have defined four subpopulations of oral cancer cells. These subpopulations showed stochastic inter-conversions as well as a strict non-interconvertible transition among them to acheive heterogeneity. Importantly, transcriptional states of each subpopulations indicated a clinically relevant signatures for patient prognosis. Also, we observed interconversions of these subpopulations in response to Cisplatin to accumulate drug-tolerant cell state, as rapid and reversible strategy to respond to chemotherapy induced stress. Thus, the characteristics of described phenotypic subgroups may be translated to the clinic for estimating the extent of intratumoral heterogeneity in oral cancer patients.

cancer biology↗

bioRxiv2021

E156/G and Arg158, Phe-157/del mutation in NTD of spike protein in B.1.167.2 lineage of SARS-CoV-2 leads to immune evasion through antibody escape

Emerging variants of SARS-CoV-2 with better immune escape mechanisms and higher transmissibility remains a persistent threat across the globe. B.1.617.2 (Delta) variant was first emerged from Maharashtra, India in December, 2020. This variant is classified to be a major cause and concern of the second wave of COVID-19 in India. In the present study, we explored the genomic and structural basis of this variant through computational analysis, protein modelling and molecular dynamics (MD) simulations approach. B.1.617.2 variant carried E156G and Arg158, Phe-157/del mutations in NTD of spike protein. These mutations in N-terminal domain (NTD) of spike protein of B.1.617.2 variant revealed more rigidity and reduced flexibility compared to spike protein of Wuhan isolate. Further, docking and MD simulation study with 4A8 monoclonal antibody which was reported to bind NTD of spike protein suggested reduced binding of B.1.617.2 spike protein compared to that of spike protein of Wuhan isolate. The results of the present study demonstrate the possible case of immune escape and thereby fitness advantage of the new variant and further warrants demonstration through experimental evidence. Our study identified the probable mechanism through which B.1.617.2 variant is more pathogenically evolved with higher transmissibility as compared to the wild-type. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/447321v3_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@b92cborg.highwire.dtl.DTLVardef@1d261f7org.highwire.dtl.DTLVardef@11da73eorg.highwire.dtl.DTLVardef@1cef6ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2021

Multiview super-resolution microscopy

We enhance the performance of confocal microscopy over imaging scales spanning tens of nanometers to millimeters in space and milliseconds to hours in time, improving volumetric resolution more than 10-fold while simultaneously reducing phototoxicity. We achieve these gains via an integrated, four-pronged approach: 1) developing compact line-scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; 2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; 3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labeled, thick samples; 4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than twenty distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae, and adults; myoblasts in Drosophila wing imaginal disks; and mouse renal, esophageal, cardiac, and brain tissues.

bioengineering↗

bioRxiv2021

Integrated phospho-proteogenomic and single-cell transcriptomic analysis of meningiomas establishes robust subtyping and reveals subtype-specific immune invasion

Meningiomas are the most frequent primary intracranial tumors. They can follow a wide clinical spectrum from benign to highly aggressive clinical course. No specific therapy exists for refractory cases or cases not amenable to resection and radiotherapy. Identification of risk of recurrence and malignant transformation for the individual patients is challenging. However, promising molecular markers and prognostic subgrouping by DNA methylation are emerging. Still, the biological underpinnings of these diagnostic subgroups are elusive, and, consequently, no novel therapeutic options arise thereof. Here we establish robust subgroups across the full landscape of meningiomas, consistent through DNA methylation, mutations, the transcriptomic, proteomic and phospho-proteomic level. Pronounced proliferative stress and DNA damage repair signals in malignant cells and in clusters exclusive to recurrent tumors are in line with their higher mitotic activity, but also provide an explanation for the accumulation of genomic instability in anaplastic meningiomas. Although homozygous deletion of CDKN2A/B is a diagnostic marker of high-grade meningioma, the expression of its gene product increased from low to non-deleted high-grade cases. Differences between subgroups in lymphocyte and myeloid cell infiltration, representing a majority of tumor mass in low-grade NF2 tumors, could be assigned to cluster-specific interaction with tumor cells. Activation to a more proinflammatory phenotype and decreased infiltration of myeloid cells in high-grade cases correlated with lower expression of CSF1, located on chromosome arm 1p, whose deletion is known as prognostic marker, with no proposed mechanism before. Our results demonstrate a robust molecular subclassification of a tumor type across multiple layers, provide insight into heterogeneous growth dynamics despite shared pathognomonic mutations, and highlight immune infiltration modulation as a novel target for meningioma therapy.

genomics↗

bioRxiv2021

A Pan-Cancer Census of Dominant Tumor Immune Archetypes

Cancers display significant heterogeneity with respect to tissue of origin, driver mutations and other features of the surrounding tissue. It is likely that persistent tumors differentially engage inherent patterns-here Archetypes-of the immune system, to both benefit from a tumor immune microenvironment (TIME) and to disengage tumor-targeting. To discover dominant immune system archetypes, the Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub compartments uncovered archetypes that exist across indications. These Immune composition-based archetypes differentiate tumors based upon unique immune and tumor gene-expression patterns. Archetypes discovered this way also tie closely to well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune infiltration and provides a rational path forward to learn how to modulate these patterns to improve therapy. O_FIG O_LINKSMALLFIG WIDTH=182 HEIGHT=200 SRC="FIGDIR/small/441344v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@55a5ccorg.highwire.dtl.DTLVardef@f8205corg.highwire.dtl.DTLVardef@1480b8org.highwire.dtl.DTLVardef@eb94e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3 splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

cancer biology↗

bioRxiv2021

Measurement of Klebsiella Intestinal Colonization Density to Assess Infection Risk

BackgroundKlebsiella pneumoniae and closely related species K. variicola and K. quasipneumoniae are common causes of healthcare-associated infections, and patients frequently become infected with their intestinal colonizing strain. To assess the association between Klebsiella colonization density and subsequent infections, a case-control study was performed. MethodsA multiplex qPCR assay was developed and validated to quantify Klebsiella (K. pneumoniae, K. variicola, and K. quasipneumoniae combined) relative to total bacterial DNA copies in rectal swabs. Cases of Klebsiella infection were identified based on clinical definitions and having a clinical culture isolate and preceding or co-incident colonization isolate with the same wzi capsular sequence type. Controls were colonized patients without subsequent infection and were matched 2:1 to cases based on age, sex, and rectal swab collection date. Quantitative PCR (qPCR) from rectal swab samples was used to measure the association between relative abundance (RA) of Klebsiella and subsequent infections. ResultsKlebsiella RA by qPCR highly correlated with 16S sequencing ({rho}=0.79; P <.001). The median Klebsiella RA in the study group was 2.6% (interquartile range (IQR) 0.1-22.5, n=238), and was higher in cases (15.7%, IQR 0.93-52.6%, n=83) than controls (1.01%, IQR 0.02-12.8%; n=155; P <0.0001). After adjusting for multiple clinical covariates using inverse probability of treatment weighting, subjects with a Klebsiella RA > 22% had a 2.87-fold (1.64-5.03, P =0.0003) increased odds of infection compared to those with lower colonization density levels. ConclusionsMeasurement of colonization density by qPCR could represent a novel approach to identify hospitalized patients at risk for Klebsiella infection. ImportanceColonization by bacterial pathogens often precedes infection, and offers a window of opportunity to prevent these infections. Klebsiella colonization is significantly and reproducibly associated with subsequent infection, however factors that enhance or mitigate this risk in individual patients are unclear. This study developed an assay to measure the density of Klebsiella colonization, relative to total fecal bacteria, in rectal swabs from hospitalized patients. Applying this assay to 238 colonized patients, high Klebsiella density defined as >22% of total bacteria, was significantly associated with subsequent infection. Based on widely available polymerase chain reaction (PCR) technology, this type of assay could be deployed in clinical laboratories to identify patients at increased risk of Klebsiella infections. As novel therapeutics are developed to eliminate pathogens from the gut microbiome, a rapid Klebsiella colonization density assay could identify patients who would benefit from this type of infection prevention interventions.

microbiology↗

bioRxiv2021

A single-cell based precision medicine approach using glioblastoma patient-specific models

Glioblastoma (GBM) is a heterogeneous tumor made up of cell states that evolve over time. Here, we modeled tumor evolutionary trajectories during standard-of-care treatment using multimodal single-cell analysis of a primary tumor sample, corresponding mouse xenografts subjected to standard of care therapy, and recurrent tumor at autopsy. We mined the multimodal data with single cell SYstems Genetics Network AnaLysis (scSYGNAL) to identify a network of 52 regulators that mediate treatment-induced shifts in xenograft tumor-cell states that were also reflected in recurrence. By integrating scSYGNAL-derived regulatory network information with transcription factor accessibility deviations derived from single-cell ATAC-seq data, we developed consensus networks that regulate subpopulations of primary and recurrent tumor cells. Finally, by matching targeted therapies to active regulatory networks underlying tumor evolutionary trajectories, we provide a framework for applying single-cell-based precision medicine approaches in a concurrent, neo-adjuvant, or recurrent setting. SummaryInference of mechanistic drivers of therapy-induced evolution of glioblastoma at single cell resolution using RNA-seq and ATAC-seq from patient samples and model systems undergoing standard-of-care treatment informs strategy for identification of tumor evolutionary trajectories and possible cell state-directed therapeutics.

systems biology↗

bioRxiv2021

Gut microbiome features are associated with sepsis onset and outcomes

BackgroundEpidemiologic studies have linked antibiotic exposure to subsequent sepsis, suggesting that microbiome disruption may be in the causal pathway and an independent risk factor. This study tests whether variation in the gut microbiota associates with risk of sepsis onset and its outcomes. MethodsUsing a validated surveillance definition, patients with an archived rectal swab from intensive care and hematology units were screened for sepsis. After confirmation by chart review, cases were matched to controls in a 1:2 ratio based on age, gender, and collection date. Relative taxon abundance was measured by sequence analysis of 16S rRNA gene amplicons; total bacterial abundance was measured by qPCR of the 23S rRNA gene. Conditional logistic regression identified clinical and microbiota variables associated with sepsis. ResultsThere were 103 sepsis cases matched to 206 controls. In a final model adjusting for exposure to broad-spectrum antibiotics and indwelling vascular catheters, high relative abundance (RA) of Enterococcus (Odds Ratio (OR) 1.36 per 10% increase, P=.016) and high total bacterial abundance (OR 1.50 per 10-fold increase in 23S copies/L, P =.001) were independently associated with sepsis. Decreased RA of butyrate-producing bacteria also independently associated with sepsis (OR 1.20 for 10% decrease in RA, P =.041), and mortality in unadjusted analysis (OR=1.47 for 10% decrease in RA, P=.034). ConclusionsThis study indicates that the microbiota is altered at sepsis onset. The decreased RA of butyrate-producing bacteria in sepsis also associates with mortality, suggesting a therapeutic role for prebiotics and probiotics in the prevention and treatment of sepsis. ImportanceEarly detection of patients at risk for sepsis could enable interventions to prevent or rapidly treat this life-threatening condition. Prior antibiotic treatment is associated with sepsis, suggesting that disruption of the bacterial population in the gut (the intestinal microbiome) could be an important step leading to disease. To investigate this theory, we matched hospitalized patients with and without sepsis and characterized the patients microbiomes close to or at onset of sepsis. We found that several microbiome alterations, including having more total bacteria in the gut was associated with onset, regardless of prior antibiotic treatment. This signature of microbiome disruption brings us closer to identifying the biological causes of sepsis and could be used to develop new diagnostic tests to identify patients at risk of sepsis.

microbiology↗

bioRxiv2020

Signature morpho-electric, transcriptomic, and dendritic properties of extratelencephalic-projecting human layer 5 neocortical pyramidal neurons

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The highly distinctive morpho-electric properties of these neurons have mainly been described in rodents, where ET neurons can be labeled by retrograde tracers or transgenic lines. Similar labeling strategies are not possible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically-defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology and morphology are assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were also apparent but were often smaller than differences between cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy, but also highlight marked phenotypic divergence possibly related to functional specialization of human neocortex.

neuroscience↗

bioRxiv2020

Plasmodium falciparum guanylyl cyclase-alpha and the activity of its appended P4-ATPase domain are essential for cGMP synthesis and blood stage egress

In malaria parasites, guanylyl cyclases (GCs), which synthesise cyclic GMP (cGMP), are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here we investigate the role of Plasmodium falciparum guanylyl cyclase alpha (GC) and its associated P4-ATPase module, showing that asexual blood stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GC-null parasites cannot synthesise cGMP, or mobilise calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that ATPase activity is up stream of and linked to cGMP synthesis. Collectively, our results demonstrate that GC is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress.

molecular biology↗