Biology subjects

Goel, A.

Publications and source records attributed to Goel, A..

30 records · Page 2Linked to original sources

bioRxiv2021

Marker-free characterization of single live circulating tumor cell full-length transcriptomes

The identification and characterization of circulating tumor cells (CTCs) are important for gaining insights into the biology of metastatic cancers, monitoring disease progression, and medical management of the disease. The limiting factor that hinders enrichment of purified CTC populations is their sparse availability, heterogeneity, and altered phenotypic traits relative to the tumor of origin. Intensive research both at the technical and molecular fronts led to the development of assays that ease CTC detection and identification from the peripheral blood. Most CTC detection methods use a mix of size selection, immune marker based white blood cells (WBC) depletion, and positive enrichment antibodies targeting tumor-associated antigens. However, the majority of these methods either miss out on atypical CTCs or suffer from WBC contamination. Single-cell RNA sequencing (scRNA-Seq) of CTCs provides a wealth of information about their tumors of origin as well as their fate and is a potent method of enabling unbiased identification of CTCs. We present unCTC, an R package for unbiased identification and characterization of CTCs from single-cell transcriptomic data. unCTC features many standard and novel computational and statistical modules for various analysis tasks. These include a novel method of scRNA-Seq clustering, named Deep Dictionary Learning using K-means clustering cost (DDLK), expression based copy number variation (CNV) inference, and combinatorial, marker-based verification of the malignant phenotypes. DDLK enables robust segregation of CTCs and WBCs in the pathway space, as opposed to the gene expression space. We validated the utility of unCTC on scRNA-Seq profiles of breast CTCs from six patients, captured and profiled using an integrated ClearCell(R) FX and PolarisTM workflow that works by the principles of size-based separation of CTCs and marker based WBC depletion.

bioinformatics↗

bioRxiv2021

The protective effect of aspirin in colorectal carcinogenesis: a multiscale computational study from mutant evolution to age incidence curves

Aspirin intake has been shown to lead to significant protection against colorectal cancer, e.g. with an up to two-fold reduction in colorectal adenoma incidence rates at higher doses. The mechanisms contributing to protection are not yet fully understood. While aspirin is an anti-inflammatory drug and can thus influence the tumor microenvironment, in vitro and in vivo experiments have recently shown that aspirin can also have a direct effect on cellular kinetics and fitness. It reduces the rate of tumor cell division and increases the rate of cell death. The question arises whether such changes in cellular fitness are sufficient to significantly contribute to the epidemiologically observed protection. To investigate this, we constructed a class of mathematical models of in vivo evolution of advanced adenomas, parameterized it with available estimates, and calculated population level incidence. Fitting the predictions to age incidence data revealed that only a model that included colonic crypt competition can account for the observed age-incidence curve. This model was then used to predict modified incidence patterns if cellular kinetics were altered as a result of aspirin treatment. We found that changes in cellular fitness that were within the experimentally observed ranges could reduce advanced adenoma incidence by a sufficient amount to account for age incidence data in aspirin-treated patient cohorts. While the mechanisms that contribute to the protective effect of aspirin are likely complex and multi-factorial, our study demonstrates that direct aspirin-induced changes of tumor cell fitness can significantly contribute to epidemiologically observed reduced incidence patterns.

cancer biology↗

bioRxiv2021

Development of a rapid LC-MS/MS method for the simultaneous quantification of various flavonoids and phytohormones extracted from Medicago Truncatula leaves

Flavonoids are small metabolites of plants, which are involved in the regulation of plant development as well as defence against pathogens. Quantitation of these flavonoids in plant samples is highly important and essential in the food and herbal industry. Hence, robust, reliable and sensitive methods are required for the analysis of these compounds in plant samples. In the present study, a high performance liquid chromatographic-tandem mass spectrometric (HPLC- ESI-MS/MS) method was developed and validated for the determination of nine flavonoids, including Liquiritigenin, Naringenin, Genistein, Daidzein, Formononetin, Biochanin A, 2-hydroxy formononetin, 2-methoxyformononetin, Medicarpin, and two phytohormones, Salicylic acid and Jasmonic acid, in Medicago truncatula. The analytes were separated by means of C-18, reversed-phase chromatography and detected using QTRAP mass spectrometer. Molecules were quantified using different transitions in positive and negative ion modes simultaneously in 12 minutes. The on-column limit of detection of all the analytes was as low as 0.03 pg, whereas the limit of quantification of all the compounds was observed upto 0.1 pg levels. Further the method was also validated in terms of specificity, linearity (r2> 0.99), average recovery (90.6 - 110.3%), accuracy (RE% [≤] 3%) and precision (RSD% [≤] 3%). As a proof of concept, the developed method was successfully used for the quantitation of these flavonoids and phytohormones from leaf extract of Medicago truncatula.

biochemistry↗

bioRxiv2021

Long term consumption of green tea EGCG enhances healthspan and lifespan in mice bymitigating multiple aspects of cellular senescence in mitotic and post-mitotic tissues, gutdysbiosis and immunosenescence

Cellular senescence is emerging as the causal nexus of aging, and its potential modulators present an effective strategy to counter age-related morbidity. The current study profiled the extent of cellular senescence in different organs of mice at four different time-points of lifespan, and explored the influence of epigallocatechin gallate (EGCG) consumption in impacting multiple aspects of aging biology. We report that adipose and intestinal tissues are highly vulnerable to cellular senescence as evident by age-associated increase in DNA damage response, activation of cell cycle inhibitors (p53/p21) and induction of SASP (p38MAPK/NF-{kappa}B/Cox-2). Further, a distinct modulation of nutrient signaling pathway mediators (AMPK/Akt/SIRT3 and 5), and a decrease in autophagy effectors was also observed in aging animals. Systemic inflamm-aging markers (TNF-/IL-l{beta}) and splenic CD4/CD8 ratio increased with age, while NK cell population decreased. Metagenomic analyses revealed age-related decrease in the diversity of microbial species while an increase in the abundance of various pathogenic bacterial genera was also observed. Long term EGCG consumption enhanced lifespan of animals by attenuating markers of DNA damage, cell cycle inhibitors and SASP in adipose, intestine and liver tissue. Mechanistically, EGCG inhibited the activation of AMPK and Akt and enhanced mitochondrial SIRT3 and SIRT5 expression, as well as autophagic response in adipose and intestinal tissues. Systemic presence of inflamm-aging markers decreased while expression of T cell immune response regulator CD69 increased in EGCG fed animals. EGCG also improved age-related decrease in the diversity of microbial species and suppressed the growth of pathogenic microbes. In short, our results provide compelling evidence that post-mitotic adipose tissue is a major site of cellular senescence and SASP activation, and that chronic EGCG consumption can influence several aspects of aging and senescence resulting in improved organismal healthspan and lifespan.

molecular biology↗

bioRxiv2020

A genetic screen identifies dreammist as a regulator of sleep

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene, dreammist (dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed dmist gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of either fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since atpa1a3a and dmist mutants have elevated intracellular Na+ levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+ pump function modulates neuronal excitability to maintain normal sleep behaviour. Significance statementSleep is an essential behavioral state, but the genes that regulate sleep and wake states are still being uncovered. A viral insertion screen in zebrafish identified a novel sleep mutant called dreammist, in which a small, highly-conserved transmembrane protein is disrupted. The discovery of dreammist highlights the importance of a class of small transmembrane-protein modulators of the sodium pump in setting appropriate sleep duration.

neuroscience↗

bioRxiv2020

Cellular heterogeneity-adjusted clonal methylation (CHALM) provides better prediction of gene expression

Promoter DNA methylation is a well-established mechanism of transcription repression, but its global correlation with gene expression is surprisingly weak. This weak correlation is due in part to the failure of current methylation quantification methods to consider the heterogeneity among sequenced bulk cells. Here, we introduce Cell Heterogeneity-Adjusted cLonal Methylation (CHALM) as a novel methylation quantification method. CHALM better explains the functional consequences of DNA methylation, such as its correlations with gene expression and H3K4me3. When applied to different methylation datasets, the CHALM method has uniquely detected differentially methylated genes that exhibit distinct biological functions supporting underlying mechanisms.

bioinformatics↗

bioRxiv2020

EZcalcium: Open Source Toolbox for Analysis of Calcium Imaging Data

Fluorescence calcium imaging using a range of microscopy approaches, such as 2-photon excitation or head-mounted miniscopes, is one of the preferred methods to record neuronal activity and glial signals in various experimental settings, including acute brain slices, brain organoids, and behaving animals. Because changes in the fluorescence intensity of genetically encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on inferring such spiking from changes in pixel intensity values across time within different regions of interest. However, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines. For decades, the only way to perform these analyses was for individual laboratories to write their own custom code. These routines were typically not well annotated and lacked intuitive graphical user interfaces (GUIs), which made it difficult for scientists in other laboratories to adopt them. Although the panorama is changing with recent tools like CaImAn, Suite2P and others, there is still a barrier for many laboratories to adopt these packages, especially for potential users without sophisticated programming skills. As 2-photon microscopes are becoming increasingly affordable, the bottleneck is no longer the hardware, but the software used to analyze the calcium data in an optimal manner and consistently across different groups. We addressed this unmet need by incorporating recent software solutions for motion correction, segmentation, signal extraction and deconvolution of calcium imaging data into an open-source, easy to use, GUI-based, intuitive and automated data analysis software, which we named EZcalcium.

neuroscience↗

bioRxiv2019

Sub-nucleosomal organization in urine cell-free DNA

Cell-free DNA (cfDNA) in urine is a promising analyte for noninvasive diagnostics. However, urine cfDNA is highly fragmented and whether characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we perform comprehensive characterization of fragmentation patterns in urine cfDNA. We show modal size and genome-wide distribution of urine cfDNA fragments are consistent with transient protection from degradation by stable intermediates of nucleosome disassembly. Genome-wide nucleosome occupancy and fragment sizes in urine cfDNA are informative of cell of origin and renal epithelial cells are amongst the highest contributors in urine. Compared to a nucleosome occupancy map based on control urine samples, we observe a higher fraction of fragments with aberrant ends in cancer patients, distinguishing cancer samples with an area under the curve of 0.89. Our results demonstrate sub-nucleosomal organization in urine cfDNA and are proof-of-principle that genome-wide fragmentation analysis of urine cfDNA can enable cancer diagnostics.

genomics↗

bioRxiv2018

Polycomb Group protein EZH2-mediated transcriptional repression of microRNA-338/-421 drives SPINK1-positive prostate cancer

PurposeSerine Peptidase Inhibitor, Kazal type-1 (SPINK1) overexpression defines the second most recurrent and aggressive prostate cancer (PCa) subtype. However, the underlying molecular mechanism and pathobiology of SPINK1 in PCa remains largely unknown.\n\nExperimental DesignMicroRNA-prediction tools were employed to examine the SPINK1-3UTR for miRNAs binding. Luciferase reporter assays were performed to confirm the SPINK1-3UTR binding of shortlisted miR-338-5p/miR-421. Further, miR-338-5p/-421 overexpressing cancer cells (SPINK1-positive) were evaluated for oncogenic properties using cell-based functional assays and mice xenograft model. Global gene expression profiling was performed to unravel the biological pathways altered by miR-338-5p/-421. Immunohistochemistry and RNA in-situ hybridization was carried-out on PCa patients tissue microarray for SPINK1 and EZH2 expression respectively. Chromatin immunoprecipitation assay was performed to examine EZH2 occupancy on the miR-338-5p/-421 regulatory regions. Bisulfite sequencing and methylated DNA-immunoprecipitation was performed on PCa cell lines and patients specimens.\n\nResultsWe established a critical role of miRNA-338-5p/-421 in post-transcriptional regulation of SPINK1. Ectopic expression of miRNA-338-5p/-421 in SPINK1-positive PCa cells abrogate oncogenic properties including cell-cycle progression, stemness and drug resistance, and show reduced tumor burden and distant metastases in mice model. Importantly, we show SPINK1-positive PCa patients exhibit increased EZH2 expression, suggesting its role in miRNA-338-5p/-421 epigenetic silencing. Furthermore, presence of CpG dinucleotide DNA methylation marks on the regulatory regions of miR-338-5p/-421 in SPINK1-positive PCa cells and patients specimens confirms epigenetic silencing.\n\nConclusionOur findings revealed that miRNA-338-5p/-421 are epigenetically silenced in SPINK1-positive PCa, while restoring the expression of these miRNAs using epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis.\n\nTRANSLATIONAL IMPACTWe establish a regulatory model involving the functional interplay between SPINK1, miRNA-338-5p/miRNA-421 and EZH2, thereby, revealing hitherto unknown mechanism of SPINK1 up-regulation in SPINK1-positive subtype. Our findings provide a strong rationale for the development of potential therapeutic strategies for SPINK1-positive malignancies. We demonstrate that restoring miRNA-338-5p/miRNA-421 expression using epigenetic drugs including DNMTs inhibitors in combination with HDACs or HKMTs inhibitors or miRNA synthetic mimics in SPINK1-positive prostate cancer abrogate SPINK1-mediated oncogenicity. The major findings of this study will not only advance the prostate cancer field, but will also be valuable for treatment and disease management of other SPINK1-positive malignancies.

cancer biology↗

bioRxiv2018

Two Isoforms of the Guanine Nucleotide Exchange Factor, Daple/CCDC88C Cooperate as Tumor Suppressors

Previously Aznar et al., showed that Daple enables Wnt/Frizzled receptors to transactivate trimeric G proteins during non-canonical Wnt signaling via a novel G-protein binding and activating (GBA) motif. By doing so, Daple serves as a double-edged sword; earlier during oncogenesis it suppresses neoplastic transformation and tumor growth, but later it triggers epithelial messenchymal transition (EMT). We have identified and characterized two isoforms of the human Daple/CCDC88c gene. While both isoforms cooperatively suppress tumor growth via their GBA motif, only the full-length transcript triggers EMT and invasion. Aspirin suppresses the full-length transcript and protein but upregulates the short isoform. Both isoforms are suppressed during colon cancer progression, and their reduced expression carries additive prognostic significance. These findings provide insights into the opposing roles of Daple during cancer progression and define the G protein regulatory GBA motif as one of the minimal modules essential for Daples role as a tumor suppressor.

cancer biology↗

bioRxiv2017

Impaired perceptual learning in Fragile X syndrome is mediated by parvalbumin neuron dysfunction in V1 and is reversible.

Atypical sensory processing is a core characteristic in autism spectrum disorders1 that negatively impacts virtually all activities of daily living. Sensory symptoms are predictive of the subsequent appearance of impaired social behavior and other autistic traits2, 3. Thus, a better understanding of the changes in neural circuitry that disrupt perceptual learning in autism could shed light into the mechanistic basis and potential therapeutic avenues for a range of autistic symptoms2. Likewise, the lack of directly comparable behavioral paradigms in both humans and animal models currently limits the translational potential of discoveries in the latter. We adopted a symptom-to-circuit approach to uncover the circuit-level alterations in the Fmr1-/- mouse model of Fragile X syndrome (FXS) that underlie atypical visual discrimination in this disorder4, 5. Using a go/no-go task and in vivo 2-photon calcium imaging in primary visual cortex (V1), we find that impaired discrimination in Fmr1-/- mice correlates with marked deficits in orientation tuning of principal neurons, and a decrease in the activity of parvalbumin (PV) interneurons in V1. Restoring visually evoked activity in PV cells in Fmr1-/- mice with a chemogenetic (DREADD) strategy was sufficient to rescue their behavioral performance. Finally, we found that human subjects with FXS exhibit strikingly similar impairments in visual discrimination as Fmr1-/- mice. We conclude that manipulating orientation tuning in autism could improve visually guided behaviors that are critical for playing sports, driving or judging emotions.

neuroscience↗

bioRxiv2017

Glucose limitation in Lactococcus shapes a single-peaked fitness landscape exposing membrane occupancy as a constraint

A central theme in biology is to understand the molecular basis of fitness: which strategies succeed under which conditions; how are they mechanistically implemented; and which constraints shape trade-offs between alternative strategies. We approached these questions with parallel bacterial evolution experiments in chemostats. Chemostats provide a constant environment with a defined resource limitation (glucose), in which the growth rate can be controlled. Using Lactococcus lactis, we found a single mutation in a global regulator of carbon metabolism, CcpA, to confer predictable fitness improvements across multiple growth rates. In silico protein structural analysis complemented with biochemical and phenotypic assays, show that the mutation reprograms the CcpA regulon, specifically targeting transporters. This supports that membrane occupancy, rather than biosynthetic capacity, is the dominant constraint for the observed fitness enhancement. It also demonstrates that cells can modulate a pleiotropic regulator to work around limiting constraints.

genomics↗