Biology subjects

Patel, A.

Publications and source records attributed to Patel, A..

162 records · Page 9Linked to original sources

bioRxiv2019

An improved plant toolset for high-throughput recombineering

Gene functional studies often rely on the expression of a gene of interest as transcriptional and translational fusions with specialized tags. Ideally, this is done in the native chromosomal contexts to avoid potential misexpression artifacts. Although recent improvements in genome editing make it possible to directly modify the target genes in their native chromosomal location, classical transgenesis is still the preferred experimental approach chosen in most gene tagging studies because of its time efficiency and accessibility. We have developed a recombineering-based tagging system that brings together the convenience of the classical transgenic approaches and the high degree of confidence in the obtained results provided by the direct chromosomal tagging achievable by genome editing strategies. These simple and customizable recombineering toolsets and protocols allow for high-throughput generation of a variety of genetic modifications. In addition, a highly efficient recombinase-mediated cassette exchange system has been developed to facilitate the transfer of the desired sequences from a BAC clone to a transformation-compatible binary vector, expanding the use of the recombineering approaches beyond Arabidopsis. The utility of this system is demonstrated by the generation of over 250 whole-gene translational fusions and 123 Arabidopsis transgenic lines corresponding to 62 auxin-related genes, and the characterization of the translational reporter expression patterns for 14 auxin biosynthesis genes.

plant biology↗

bioRxiv2019

Functionalized microcarriers improve T cell manufacturing by facilitating migratory memory T cell production and increasing CD4/CD8 ratio

Adoptive cell therapies (ACT) using chimeric antigen receptor (CAR) T cells have shown promise in treating cancer, but manufacturing large numbers of high quality cells remains challenging. Critically, current T cell expansion technologies only partially recapitulate the in vivo microenvironment found in the human lymph nodes. In these organs, T cells expand at high cell density with autocrine/paracrine signaling, as well as signals from the extracellular matrix (ECM). Here we describe a T cell expansion system using degradable gelatin microcarriers functionalized with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), which address several of these shortcomings. We show that using this system, we can achieve approximately 2-fold greater expansion compared to functionalized magnetic beads, the current industry standard. Furthermore, carriers generated higher numbers of CCR7+CD62L+ migratory, central memory T cells and CD4+ T cells across multiple donors. Both these phenotypes have emerged as important for establishing durable and effective responses in patients receiving T cell immunotherapies. We further demonstrate that carriers can achieve greater memory cell yield compared to beads across a range of IL2 concentrations from 20 U/mL to 100 U/mL. These differences were greater at lower IL2 concentrations, indicating that the carriers are more efficient. We optimized this system using a design of experiments (DOE) approach and found that the carrier concentration affects the memory cell yield in a quadratic manner, where high or low concentrations are detrimental to memory formation. Finally, we show that carriers do not hinder CAR transduction and can maintain the CD4 and memory phenotype advantages in CAR-transduced T cells.

bioengineering↗

bioRxiv2019

Cooperating lineage factors SOX2 and BRN2 govern the transcriptional program of the neural subtype of lung squamous cell carcinoma

Molecular characterization of lung squamous cell carcinoma (LUSC), a major subtype of lung cancer, has not sufficiently improved its non-stratified treatment strategies over decades. Accumulating evidence suggests that lineage-specific transcriptional regulators control differentiation states during cancer evolution, and underlie their distinct biological behaviors. In this study, by investigating the super-enhancer landscape of LUSC, we identified a previously undescribed neural subtype defined by Sox2 and a neural lineage factor Brn2, as well as the classical LUSC subtype defined by Sox2 and its classical squamous partner p63. Robust protein-protein interaction and genomic co-occupancy of Sox2 and Brn2, in place for p63 in the classical LUSC, indicated their transcriptional cooperation imparting this unique lineage state in the neural LUSC. Forced expression of p63 downregulated Brn2 in the neural LUSC cells and invoked the classical LUSC lineage with more squamous/epithelial features, which were accompanied by increased activities of ErbB/Akt and MAPK-ERK pathways suggesting differential dependency. Collectively, our data demonstrate heterogeneous cell lineage states of LUSC featured by Sox2 cooperation with Brn2 or p63, for which distinct therapeutic approaches may be warranted.

cancer biology↗

bioRxiv2019

Gating Mechanism of β-Ketoacyl-ACP Synthases

Formation of carbon-carbon bonds via {beta}-ketoacyl-acyl carrier protein (ACP) synthases (KS), are key reactions during de novo fatty acid and polyketide biosynthesis. KSs recognize multiple ACPs and choreograph ping-pong mechanisms often in an iterative fashion. Therefore, KSs must limit non-productive protein-protein interactions (PPIs) to achieve high degrees of reaction fidelity. To better understand the stereochemical features governing substrate discrimination during ACP*KS PPIs, we determined x-ray crystal structures complemented by molecular dynamic simulations of E. coli AcpP in complex with the elongating KSs, FabF and FabB. Covalently trapped substrate analogs were used to interrogate critical catalytic events accompanying carbon-carbon bond formation revealing a previously unknown gating mechanism during the binding and delivery of acyl-AcpPs. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features generally in elongating KSs.

biochemistry↗

bioRxiv2019

The Glycolytic Protein Phosphofructokinase Dynamically Relocalizes into Subcellular Compartments with Liquid-like Properties in vivo

While much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. Here we built a hybrid microfluidic-hydrogel device for use in Caenorhabditis elegans to systematically examine and quantify the dynamic subcellular localization of the rate-limiting enzyme of glycolysis, phosphofructokinase-1/PFK-1.1. We determine that endogenous PFK-1.1 localizes to distinct, tissue-specific subcellular compartments in vivo. In neurons, PFK-1.1 is diffusely localized in the cytosol, but capable of dynamically forming phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in the dispersion of PFK-1.1 in the cytosol, indicating that PFK-1.1 reversibly organizes into biomolecular condensates in response to cues within the cellular environment. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Prolonged conditions of energy stress during sustained hypoxia alter the biophysical properties of PFK-1.1 in vivo, affecting its viscosity and mobility within phase-separated condensates. PFK-1.1s ability to form tetramers is critical for its capacity to form condensates in vivo, and heterologous self-association domain such as cryptochrome 2 (CRY2) is sufficient to constitutively induce the formation of PFK-1.1 condensates. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically compartmentalize in vivo to specific subcellular compartments in response to acute energy stress via multivalency as phase-separated condensates.

cell biology↗

bioRxiv2019

Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the -subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2 I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to <2% for the 3D7 parental line (P = 0.0011 for each clone). In immunoprecipitation and localisation studies of HA-tagged AP-2, we identified interacting partners including AP-2, AP-1/2{beta}, AP-2{sigma} and a kelch-domain protein encoded on chromosome 10 of P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

microbiology↗

bioRxiv2019

A new immunotherapeutic concept for pancreatic cancer using childhood vaccine recall antigens and Listeria

Pancreatic ductal adenocarcinoma is highly metastatic, poorly immunogenic, and immune suppression prevents T cell activation in the tumor microenvironment. We developed a microbial-based immunotherapeutic concept for selective delivery of a highly immunogenic tetanus toxoid protein (TT856-1313), into tumor cells by attenuated Listeria monocytogenes, and reactivation of pre-existing TT-specific memory T cells (generated during childhood) to kill infected tumor cells. Thus, TT here functions as an alternative for neoantigens. Treatment of KPC mice with Listeria-TT resulted in TT accumulation in tumors and inside tumor cells, and attraction of predominantly TT-specific memory CD4 T cells. Moreover, gemcitabine (GEM) combined with Listeria-TT significantly improved the migration of CD4 T cells into tumors and the production of perforin and granzyme B, turning cold tumors into immunological hot tumors. In vivo depletion of T cells in Listeria-TT+GEM-treated mice demonstrated CD4 T cell-mediated eradication of tumors and metastases (Mann-Whitney p<0.05). In addition, peritumoral lymph node like structures (LNS) were observed in close contact with the pancreatic tumors displaying CD4 T cells and CD8 T cells of KPC mice treated with Listeria-TT or Listeria-TT+GEM. The production of perforin and granzyme B was observed in LNS of KPC mice that received Listeria-TT+GEM. This combination not only reduced tumor burden (80%) and metastases (87%) significantly (p<0.05, Mann-Whitney), but also improved the survival time of KPC mice with advanced pancreatic cancer substantially (Mantel-Cox p<0.0001). Our results unveil new mechanisms of Listeria and GEM improving immunotherapy for PDAC.

immunology↗

bioRxiv2019

Rapid and iterative genome editing in the zoonotic malaria parasite Plasmodium knowlesi: New tools for P. vivax research

Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.

microbiology↗

bioRxiv2018

Using DeepLabCut for 3D markerless pose estimation across species and behaviors

Noninvasive behavioral tracking of animals during experiments is crucial to many scientific pursuits. Extracting the poses of animals without using markers is often essential for measuring behavioral effects in biomechanics, genetics, ethology & neuroscience. Yet, extracting detailed poses without markers in dynamically changing backgrounds has been challenging. We recently introduced an open source toolbox called DeepLabCut that builds on a state-of-the-art human pose estimation algorithm to allow a user to train a deep neural network using limited training data to precisely track user-defined features that matches human labeling accuracy. Here, with this paper we provide an updated toolbox that is self contained within a Python package that includes new features such as graphical user interfaces and active-learning based network refinement. Lastly, we provide a step-by-step guide for using DeepLabCut.

neuroscience↗

bioRxiv2018

Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential and conserved mediator of epithelial fusion during vertebrate embryogenesis.

Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including strong evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that Netrin-1 (NTN1) is precisely expressed in the chick fissure margin at the fusion plate but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and fish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have a major role in palate fusion. Our data reveal that NTN1 is a new locus for human coloboma and other multi-system developmental fusion defects, and that chick OFC is a powerful model for epithelial fusion research.

developmental biology↗

bioRxiv2018

C/VDdb: a multi-omics expression profiling database for a knowledge-driven approach in cardiovascular disease (CVD)

The cardiovascular disease (C/VD) database is an integrated and clustered information resource that covers multi-omic studies (microRNA, genomics, proteomics and metabolomics) of cardiovascular-related traits with special emphasis on coronary artery disease (CAD). This resource was built by mining existing literature and public databases and thereafter manual biocuration was performed. To enable integration of omic data from distinct platforms and species, a specific ontology was applied to tie together and harmonise multi-level omic studies based on gene and protein clusters (CluSO) and mapping of orthologous genes (OMAP) across species.\n\nCAD continues to be a leading cause of death in the population worldwide, and it is generally thought to be an age-related disease. However, CAD incidence rates are now known to be highly influenced by environmental factors and interactions, in addition to genetic determinants. With the complexity of CAD aetiology, there is a difficulty in research studies to elucidate general elements compared to other cardiovascular diseases.\n\nData from 92 studies, covering 13945 molecular entries (4353 unique molecules) is described, including data descriptors for experimental setup, study design, discovery-validation sample size and associated fold-changes of the differentially expressed molecular features (p-value<0.05). A dedicated interactive web interface, equipped with a multi-parametric search engine, data export and indexing menus are provided for a user-accessible browsing experience.\n\nThe main aim of this work was the development of a data repository linking clinical information and molecular differential expression in several CVD-related traits from multi-omics studies (genomics, transcriptomics, proteomics and metabolomics). As an example case of how to query and identify data sets within the database framework and concomitantly demonstrate the database utility, we queried CAD-associated studies and performed a systems-level integrative analysis.\n\nURL: www.padb.org/cvd

systems biology↗

bioRxiv2018

TEsmall identifies small RNAs associated with targeted inhibitor resistance in melanoma

MicroRNAs (miRNAs) are small 21-22nt RNAs that act to regulate the expression of mRNA target genes through direct binding to mRNA targets. While miRNAs typically dominate small RNA transcriptomes, many other classes are present including tRNAs, snoRNAs, snRNAs, Y-RNAs, piRNAs, and siRNAs. Interactions between processing machinery and targeting networks of these various small RNA classes remains unclear, largely because these small RNAs are typically analyzed separately. Here we present TEsmall, a tool that allows for the simultaneous processing and analysis of small RNAs from each annotated class in a single integrated workflow. The pipeline begins with raw fastq reads and proceeds all the way to producing count tables formatted for differential expression. Several interactive charts are also produced to look at overall distributions in length and annotation classes. We next applied the TEsmall pipeline to small RNA libraries generated from melanoma cells responding to targeted inhibitors of the MAPK pathway. Targeted oncogene inhibitors have emerged as way to tailor cancer therapies to the particular mutations present in a given tumor. While these targeted strategies are typically effective for short intervals, the emergence of resistance is extremely common, limiting the effectiveness of single-agent therapeutics and driving the need for a better understanding of resistance mechanisms. Using TEsmall, we identified several microRNAs and other small RNA classes that are enriched in inhibitor resistant melanoma cells in multiple melanoma cell lines and may be able to serve as markers of resistant populations more generally.

genomics↗

bioRxiv2018

Azithromycin reduces systemic inflammation and provides survival benefit in murine model of polymicrobial sepsis

Sepsis is a life threatening systemic inflammatory condition triggered as a result of excessive host immune response to infection. In the past, drugs modulating immune reactions have demonstrated protective effect in sepsis. Azithromycin (macrolide antibiotic) with immunomodulatory activity was therefore evaluated in combination with ceftriaxone in a more clinically relevant murine model of sepsis induced by caecal ligation and puncture (CLP). First, mice underwent CLP and 3 h later were administered with vehicle, sub-effective dose of ceftriaxone (100 mg/kg, subcutaneous) alone or in combination with immunomodulatory dose of azithromycin (100 mg/kg, intraperitoneal). Survival was then monitored for 5 days. Parameters like body temperature, blood glucose, total white blood cell count, plasma glutathione (GSH), plasma and lung myeloperoxidase (MPO) as well as cytokine (interleukin IL-6, IL-1{beta}, tumor necrosis factor-) levels along with bacterial load in blood, peritoneal fluid and lung homogenate were measured 18 h after CLP challenge. Combination group significantly improved the survival of CLP mice. It attenuated the elevated levels of inflammatory cytokines and MPO in plasma and lung tissue and increased the body temperature, blood glucose and GSH which were otherwise markedly decreased in CLP mice. Ceftriaxone exhibited significant reduction of bacterial count in blood, peritoneal fluid and lung homogenate, while co-administration of azithromycin did not further reduce it. This confirms that survival benefit by azithromycin was due to immunomodulation and not by its antibacterial action. Findings of this study indicate that azithromycin in combination with ceftriaxone could exhibit clinical benefit in sepsis.

pharmacology and toxicology↗

bioRxiv2017

SAKE (Single-cell RNA-Seq Analysis and Klustering Evaluation) Identifies Markers of Resistance to Targeted BRAF Inhibitors in Melanoma Cell Populations

Single-cell RNA-Seqs (scRNA-Seq) unprecedented cellular resolution at a genome wide scale enables us to address questions about cellular heterogeneity that are inaccessible using methods that average over bulk tissue extracts. However, scRNA-Seq datasets also present additional challenges such as high transcript dropout rates, stochastic transcription events, and complex population substructures. Here, we present SAKE (Single-cell RNA-Seq Analysis and Klustering Evaluation): a robust method for scRNA-Seq analysis that provides quantitative statistical metrics at each step of the scRNA-Seq analysis pipeline including metrics for: the determination of the number of clusters present, the likelihood that each cell belongs to a given cluster, and the association of each gene marker in determining cluster membership. Comparing SAKE to multiple single-cell analysis methods shows that most methods perform similarly across a wide range cellular contexts, with SAKE outperforming these methods in the case of large complex populations. We next applied the SAKE algorithms to identify drug-resistant cellular populations as human melanoma cells respond to targeted BRAF inhibitors. Single-cell RNA-Seq data from both the Fluidigm C1 and 10x Genomics platforms were analyzed with SAKE to dissect this problem at multiple scales. Data from both platforms indicate that BRAF inhibitor resistant cells can emerge from rare populations already present before drug application, with SAKE identifying both novel and known markers of resistance. In addition, we compare integrated genomic and transcriptomic markers to show that resistance can arise stochastically within multiple distinct clonal populations.

genomics↗

bioRxiv2017

Theta and alpha oscillations are traveling waves in the human neocortex

Human cognition requires the coordination of neural activity across widespread brain networks. Here we describe a new mechanism for large-scale coordination in the human brain: traveling waves of theta and alpha oscillations. Examining direct brain recordings from neurosurgical patients performing a memory task, we found that contiguous clusters of cortex in individual patients showed oscillations at specific frequencies in the range of 2 to 15 Hz. These clusters displayed spatial phase gradients, indicating that individual oscillation cycles moved across the cortex at ~0.25-0.75 m/s. We found that traveling waves were relevant behaviorally because their propagation correlated with task events and was more consistent during good performance. Traveling waves showed a correlation between propagation speed and temporal frequency, which suggests that they propagate across the cortex following principles of phase-coupled oscillatory networks. By demonstrating that theta and alpha traveling waves are widespread and behaviorally relevant, our results suggest a broad role for brain oscillations in supporting cortical connectivity by organizing neural activity across space and time.

neuroscience↗

bioRxiv2017

Ultra-sensitive mutation detection and genome-wide DNA copy number reconstruction by error corrected circulating tumour DNA sequencing

Minimally invasive circulating free DNA (cfDNA) analysis can portray cancer genome landscapes but highly sensitive and specific genetic approaches are necessary to accurately detect mutations with often low variant frequencies. We developed a targeted cfDNA sequencing technology using novel off-the-shelf molecular barcodes for error correction, in combination with custom solution hybrid capture enrichment. Modelling based on cfDNA yields from 58 patients shows that our assay, which requires 25ng of cfDNA input, should be applicable to >95% of patients with metastatic colorectal cancer. Sequencing of a 163.3 kb target region including 32 genes detected 100% of single nucleotide variants with 0.15% variant frequency in cfDNA spike-in experiments. Molecular barcode error correction reduced false positive mutation calls by 98.6%. In a series of 28 patients with metastatic colorectal cancers, 80 out of 91 (88%) mutations previously detected by tumour tissue sequencing were called in the cfDNA. Call rates were similar for single nucleotide variants and small insertions/deletions. Mutations only called in cfDNA but not detectable in matched tumour tissue included, among others, a subclonal resistance driver mutation to anti-EGFR antibodies in the KRAS gene, multiple activating PIK3CA mutations in each of two patients (indicative of parallel evolution), and TP53 mutations originating from clonal haematopoiesis. Furthermore, we demonstrate that cfDNA off-target read analysis allows the reconstruction of genome wide copy number aberration profiles from 71% of these 28 cases. This error-corrected ultra-deep cfDNA sequencing assay with a target region that can be readily customized enables broad insights into cancer genomes and evolution.

genomics↗

bioRxiv2017

The small molecule KHS101 induces bioenergetic dysfunction in glioblastoma cells through inhibition of mitochondrial HSPD1

Pharmacological inhibition of uncontrolled cell growth with small molecule inhibitors is a potential strategy against glioblastoma multiforme (GBM), the most malignant primary brain cancer. Phenotypic profiling of the neurogenic small molecule KHS101 revealed the chemical induction of lethal cellular degradation in molecularly-diverse GBM cells, independent of their tumor subtype, whereas non-cancerous brain cells remained viable. Mechanism-of-action (MOA) studies showed that KHS101 specifically bound and inhibited the mitochondrial chaperone HSPD1. In GBM but not non-cancerous brain cells, KHS101 elicited the aggregation of an enzymatic network that regulates energy metabolism. Compromised glycolysis and oxidative phosphorylation (OXPHOS) resulted in the metabolic energy depletion in KHS101-treated GBM cells. Consistently, KHS101 induced key mitochondrial unfolded protein response factor DDIT3 in vitro and in vivo, and significantly reduced intracranial GBM xenograft tumor growth upon systemic administration, without discernible side effects. These findings suggest targeting of HSPD1-dependent oncometabolic pathways as an anti-GBM therapy.

cancer biology↗

bioRxiv2017

Telomere elongation and Telomerase activity in Normal and Cancer cell lines: HEK-293, HeLa and A549

Telomerase, an eukaryotic ribonucleoprotein (RNP) complex, consists of an essential RNA template and a reverse transcriptase termed as human telomerase reverse transcriptase (hTERT). By using this reverse transcriptase enzyme, telomerase RNP maintains its telomeric length in all cancer cells and in few stem cells. As cells age, telomerase level decreases and telomere get shorten, leading to senescence. Presence of telomerase helps delay the senescence due to active production of telomere, leading to cell turning malignant and eventually cancerous. In this experiment, we would investigate the telomerase activity tested using q PCR and telomerase level using western blot in three different cell lines, HEK-293, HeLa and A549.\n\nHypothesisWhere HEK-293 cells have longer telomeres and higher telomerase activity, HeLa and A549 cell lines will have varied length of telomere but higher telomerase activity.

molecular biology↗