Biology subjects

Patel, B. K.

Publications and source records attributed to Patel, B. K..

10 recordsLinked to original sources

bioRxiv2026

Mechanistic Insights into Unfolding of the Mitochondrial Localization Sequence M1 of TDP-43 and Identification of a MAPKAPK2 Inhibitor as a potential binder of M1 for inhibition of TDP-43's Aberrant Mitochondrial Localization

Mechanistic elucidation and inhibition of the pathogenic aberrant mitochondrial localization of the RNA/DNA-binding protein, TDP-43, can help in the therapeutics of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A mitochondrial localization sequence of TDP-43, M1, is largely solvent inaccessible, therefore, how it interacts with the mitochondrial import machinery to facilitate TDP-43s transit to mitochondria is unclear. Towards this, we examined the unfolding TDP-43s N-terminal domain (NTD) that hosts M1, using equilibrium all-atom molecular dynamics (MD) simulations, and observed an early loss of the hydrogen-bonded interactions between {beta}4-{beta}5 bridge and the interactions involving residues Phe-35 and Gly-40 of M1, indicating structural lability of M1 to become solvent-accessible that may enhance its interaction with the mitochondrial receptor(s) for import. Furthermore, via virtual screening of 2,115 FDA-approved and 515,545 non-FDA-approved small molecules from ZINC15 database towards binding to M1 and inhibiting TDP-43s mitochondrial import, we identified a molecule, ZINC73240059, that was previously characterized as an inhibitor of MAP kinase-activating protein kinase 2 (MAPKAPK2). ZINC73240059 remains stably bound to M1 of NTD during MD simulations manifesting negative Gibbs free energy ({Delta}G) with significant contribution from Pro-36 of M1. Overall, ZINC73240059 can be a molecule of interest towards thwarting TDP-43s pathogenic mitochondrial localization in ALS.

biophysics↗

bioRxiv2025

Elucidating the conformational dynamics of the mitochondrial localization signal, M3, of TDP-43 and accessing potential inhibitors using molecular docking and simulation

Aberrant mitochondrial localization of the RNA/DNA-binding protein TDP-43 is implicated in amyotrophic lateral sclerosis (ALS) which may affect mitochondrial dynamics and contribute to neuronal toxicity. Inhibitors of the cytoplasmic aggregation of TDP-43 were reported previously, but their effect on the mitochondrial mis-localization of TDP-43 and the mitochondrial function remains to be investigated. Three internal peptide sequences from TDP-43, M1, M3, and M5, were found to enable TDP-43s mitochondrial localization and competitive inhibition using these peptides thwarted mitochondrial import of TDP-43 and rescued TDP-43-induced cytotoxicity to neurons. Here, we performed a virtual in silico screening of 2,115 FDA-approved small molecules for affinity against the M3 region of TDP-43 (aa: 146 - 150) that lies in the RNA recognition motif-1 (RRM1) domain. Multiple all-atom MD simulations were carried out starting with two different conformations of the tandem RRMs to understand the dynamics of the target M3 region in explicit solvent water. The analysis of the simulation trajectories suggests that the M3 region is relatively non-flexible and buried relative to the other regions of the tandem RRM1-2 domains. Cholecalciferol (Vitamin D3), from virtual screening, docked consistently with the M3 region in different docking strategies even amidst the regions poor solvent accessibility. Vitamin D3 also remained stably bound to the M3 region in most frames of four replica MD simulations, each of one microsecond. Taken together, our study proposes vitamin D3 as a potential binder to the M3 region which may inhibit the pathogenic mitochondrial mis-localization of TDP-43.

bioinformatics↗

bioRxiv2025

Intraductal Papillary Mucinous Neoplasm Cellular Plasticity linked with Repeat Element Dysregulation

BackgroundIntraductal papillary mucinous neoplasms (IPMNs) are preinvasive pancreatic lesions with a spectrum of histologic phenotypes and variable risk in progressing to invasive cancer. Aberrant repetitive element expression has been shown to be functionally linked to cell state changes in pancreatic cancer. ObjectiveThis study utilized spatial transcriptomics with customized repeat element probes to better understand the relationship of histologic subtypes, repeat element dysregulation, and molecular profiles of different cell populations in IPMN. DesignA total of 52 lesions from 18 patients with resected IPMNs of different histologies and degrees of dysplasia were analyzed with whole transcriptome spatial analysis (GeoMx). Of these, 50 lesions from 17 patients were also processed for single-cell spatial molecular imaging (CosMx). Repeat element probes for LINE1, HSATII, HERVK, and HERVH were used for GeoMx and CosMx. ResultsPancreaticobiliary-type IPMN was enriched for basal-like epithelium and infiltration of Treg cells. Intestinal-type IPMN was enriched for classical epithelium and macrophage infiltrates. Gastric-type IPMN was found to have equal basal-like and classical epithelium with a diverse immune infiltrate. Repeat RNAs were expressed at high levels across IPMN phenotypes and enriched in high-grade dysplasia. Single-cell transcriptional trajectory analysis revealed a phylogeny starting from gastric toward intestinal and pancreaticobiliary branches associated with higher-grade dysplasia and repeat RNA expression. ConclusionSpatial transcriptomics of IPMN identified a molecular continuum between histological subtypes supporting a common gastric-type origin that transitions to intestinal and pancreatobiliary phenotypes. This cell state plasticity is linked with repeat element expression that can be a potential biomarker for IPMN progression. What is already known on this topicO_LIMolecular characterization of IPMN subtypes using regional spatial transcriptomics has described the differences between histologies C_LIO_LIRepeat element expression is associated with cell state changes in pancreatic ductal neoplasm C_LI What this study addsO_LISingle cell spatial molecular imaging of IPMN subtypes reveals a cellular and molecular continuum starting from gastric histology with distinct branches to Intestinal and pancreatobiliary subtypes C_LIO_LIRepeat element expression is associated with higher grade IPMN histology C_LIO_LIRepeat element expression is elevated in the histologic and molecular continuum from gastric to intestinal and pancreatobiliary-type IPMN C_LI How this study might affect research, practice, or policyO_LIThese results support a common gastric histology origin of IPMN subtypes with different molecular trajectories towards higher grade disease C_LIO_LIOur findings suggest that repeat RNA expression can be used in conjunction with other transcriptional markers of cell state as a biomarker for IPMN progression C_LI

cancer biology↗

bioRxiv2024

Fecal Metabolite Quantitation for Rapid Assessment of the Gut Microbiome

The intestinal microbiome is composed of myriad microbial species that produce metabolites that impact host health. While loss of bacterial species and beneficial metabolites from the fecal microbiome is associated with development of a range of diseases and medical complications, there are currently no diagnostic tests that rapidly identify individuals with microbiome deficiencies. Reduced concentrations of fecal butyrate and deoxycholic acid are associated with adverse clinical outcomes and result from the loss of a subset of health-associated bacterial species. We present a rapid diagnostic test based on 3-nitrophenylhydrazine derivatization and ultrahigh-performance liquid chromatography-mass spectrometry that measures fecal butyrate and deoxycholic acid concentrations as markers of microbiome function. A matrix-matched calibration curve was developed using a simulated fecal mixture to optimize accuracy and facilitate adherence to clinical laboratory regulations. The assay showed an analytical measurement range from 4.3-3030.1 {micro}M (LLOQ = 9.75 {micro}M) for butyrate and from 0.9- 64.9 {micro}M (LLOQ = 0.9 {micro}M) for deoxycholic acid. Precision demonstrated a coefficient of variation <15% at all QC levels tested. The assay can be performed in under an hour from extraction to provision of quantitative results, enabling the rapid identification of patients with defective microbiome function.

pathology↗

bioRxiv2024

Enhanced in vitro aggregation, but not phase separation, of TDP-43 and its C-terminal fragments generate deep-blue autofluorescence.

As misfolding and aggregation of the RNA/DNA-binding protein, TDP-43, are linked to devastating TDP-43 proteinopathies like amyotrophic lateral sclerosis (ALS), distinction of the nature of the aggregated TDP-43 species being liquid-like non-pathogenic or solid-like pathogenic is important for mechanistic elucidation and therapeutic targeting. Here, we examined if in vitro enhancement of the TDP-43 aggregation can generate or enhance intrinsic deep-blue autofluorescence (dbAF) previously reported for a few other protein aggregates and whether dbAF is emitted by all or only liquid-like or solid-like TDP-43 aggregates. Using thioflavin-T fluorescence, turbidimetry, atomic force microscopy and fluorescence microscopy of Alexa Fluor-labelled protein, we first tested the in vitro enhancement of the aggregation of the full-length TDP-43 and its two C-terminal fragments (CTFs), TDP-432C (aa: 193-414) and the TDP-43-low complexity domain (LCD) (aa: 274-414). We find that presence of metal ions, Zn2+ or Mn2+, that are also linked to ALS-associated metal dyshomeostasis, or addition of a kosmotropic anion, SO42-, enhance the in vitro solid-like aggregations of the full-length TDP-43 and TDP-432C that also concurrently enhance emission of dbAF. In contrast, Alexa fluor-633-labeled-TDP-43-LCD underwent a quick phase separation into globular structures in presence of Zn2+ ions and the phase-separated species failed to emit dbAF but upon further incubation when matured into solid-like irregular, but non-amyloid nature aggregates, it emitted dbAF. Strikingly, we find that the TDP-43 aggregates of both amyloid and non-amyloid nature, but not the oligomers or the phase-separated droplets of TDP-43, manifest dbAF. Overall, the observed in vitro enhancement of aggregation leading to concurrent enhancement of dbAF can enable a label-free easy detection and may facilitate distinguishing of potentially pathogenic versus non-pathogenic TDP-43 aggregates.

biochemistry↗

bioRxiv2023

In silico and in vitro studies suggest epigallocatechin gallate (EGCG), a polyphenol in green tea, can bind and modulate the aggregation and cytotoxicity of the full-length TDP-43 protein implicated in TDP-43 proteinopathies.

Misfolding and aggregation of TDP-43 protein are implicated in several proteinopathies like ALS and FTLD. Extracellular TDP-43 is also proposed to propagate in a prion-like pathogenic manner to the neighbouring cells. Here, using turbidity and sedimentation assay, we show that a polyphenol in green tea, epigallocatechin gallate (EGCG), can inhibit the in vitro aggregation of the full-length TDP-43 protein. Furthermore, Alexa-Fluor-labelled TDP-43 protein failed to show aggregates in the presence of EGCG in fluorescence microscopy. Also, AFM imaging revealed that EGCG co-incubation with TDP-43 allows formation of only small oligomers in contrast to the larger TDP-43 aggregates formed otherwise. A physical binding of EGCG with TDP-43 was observed using triphenyl tetrazolium chloride (TTC) staining and isothermal titration calorimetry (ITC). ITC also revealed a high-affinity binding site for EGCG on TDP-43 with a Kd value of 7.8 {micro}M and a binding free energy of -6.9 kcal/mol. Furthermore, in silico molecular docking and molecular dynamic simulation (MDS) studies using different available structures of the N-terminal, RRM1-2 and C-terminal domains of TDP-43, predicted a preferable and stable binding of EGCG to the structure of the aggregation prone C-terminal domain (CTD) (PDB ID:7KWZ). Also, EGCG complexed with CTD of TDP-43 yielded a negative {Delta}G value of -20.29 kcal/mol using MM-PBSA analysis of the MDS data thereby further suggesting a stable complex formation. Also, in MDS, EGCG interacted with the amino acids Phe-313 and Ala-341 of TDP-43, which were previously projected to be important for the recruitment of monomers for the amyloid formation by CTD, thereby suggesting a possible mechanism of EGCGs inhibition of the TDP-43 aggregation. Notably, while the in vitro-made aggregates of full-length TDP-43 caused mild cytotoxicity to the HEK293 cells, the small oligomers of TDP-43 formed in presence of EGCG did not. In totality, EGCG can in vitro interact with TDP-43 and inhibit its aggregation, possibly via interaction with the amyloidogenic domain, thereby preventing it from assuming cytotoxic conformations. As EGCG is a natural molecule, it could be relevant to the therapeutic quest against the TDP-43 proteinopathies.

biochemistry↗

bioRxiv2023

Dysregulated Repeat Element Viral-like Immune Response in Hepatocellular Carcinoma

PurposeHepatocellular carcinoma (HCC) is a lethal malignancy driven by complex interactions between cancer cells, immune cells, and additional stromal cells in the tumor microenvironment (TME). The LINE1 retrotransposon is a ubiquitous repeat RNA whose de-repression leads to significant cancer cell-intrinsic and TME changes that promote aggressive tumor characteristics. We leveraged single cell spatial transcriptomic profiling to characterize the relationship between LINE1 and differences in the heterogeneous HCC TME. Experimental DesignWe applied our profiling methodology to a cohort of 23 tissue specimens collected from patients who had undergone liver resection or transplantation and validated it in a partially-overlapping similar cohort of 39 specimens using RNA in-situ hybridization (RNA-ISH). ResultsWe found that LINE1-high tumors and LINE1-high single HCC cells exhibited a de-differentiated, stem-like, and inflammatory phenotype. Furthermore, within individual tumors, LINE1 high cancer cells associated spatially with one another and excluded the larger, organized immune cell conglomerates seen in LINE1 low tumors. Finally, we found that LINE1 RNA expression correlated with worse overall survival in the larger expanded retrospective cohort. ConclusionsOur study is the first to show a clearly disorganized immune TME in HCC driven by LINE1 expression, and this observation correlated with poor survival for patients whose tumors expressed large amounts of the LINE1 repeat RNA. These results provide further evidence of how effective anti-tumor immune responses contribute to cures after definitive surgery and may lead to novel biomarkers or drug targets in HCC. TRANSLATIONAL RELEVANCEThe viral-like LINE1 retrotransposon is known to influence tumor cell state and the immune response in a variety of cancer. Here, we have used single cell spatial transcriptomic profiling to resolve repeat and coding gene RNA expression in a cohort of hepatocellular carcinoma (HCC) patients. LINE1 RNA expression in HCC tumor cells was correlated with an undifferentiated stem-like cancer state and a disorganized, sparse immune infiltrate. Using in situ hybridization on an expanded validation cohort, we noted significantly worsened survival in the LINE1 high group. Altogether, LINE1 repeat RNA is a tumor intrinsic biomarker of more aggressive features that can be used for risk stratification and a potential biomarker for response to immunotherapies that merits further investigation.

cancer biology↗

bioRxiv2020

Role of CNC1 gene in TDP-43 aggregation-induced oxidative stress-mediated cell death in S. cerevisiae model of ALS.

TDP-43 is a multi-functional ribonucleoprotein that is also found deposited as hyper-phosphorylated and ubiquitinated TDP-43 inclusions in the brain and spinal cord of the patients of the motor neuron diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Till date, how the cell death ensues is not fully deciphered although several molecular mechanisms of the TDP-43 toxicity such as impairments of endocytosis and chromatin remodelling, mis-regulations of autophagy and proteasome function, mis-localization to the mitochondria and generation of oxidative stress etc., have been proposed. A predominantly nuclear protein, Cyclin C, can regulate the oxidative stress response by affecting the transcription of stress response genes and also by translocation to the cytoplasm for the activation of the mitochondrial fragmentation-dependent cell death pathway. Using the well-established yeast model of TDP-43 aggregation and toxicity, we examined here whether upon TDP-43 aggregation, the cell survival depends on the presence of the CNC1 gene that encodes Cyclin C protein or other genes that encode proteins that function in conjunction with Cyclin C, such as the DNM1, FIS1 and MED13 genes. We found that the TDP-43 toxicity is significantly reduced in the yeast deleted for the CNC1 or DNM1 genes. Importantly, the rescue of TDP-43 toxicity in these yeast deletion backgrounds required the presence of functional mitochondria. Also, the deletion of YBH3 gene, which encodes for a protein involved in the mitochondria-dependent apoptosis, also reduced the TDP-43 toxicity. Furthermore, Cyclin C-YFP was observed to localize from the nucleus to the cytoplasm in response to the TDP-43 co-expression. Also, this cytoplasmic localization of Cyclin C was prevented by the addition of an anti-oxidant molecule, N-acetyl-cysteine. Taken together, our data suggest that Cyclin C, Dnm1 and Ybh3 proteins are important in mediating the TDP-43-induced oxidative stress-mediated cell death in the S. cerevisiae model.

genetics↗

bioRxiv2019

Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4.

TDP-43 is an RNA/DNA-binding protein of versatile physiological functions and it is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease in addition to several other implicated proteins such as mutant SOD1 and FUS etc. Cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation of TDP-43 are suggested to be important TDP-43-toxicity causing mechanisms for the ALS manifestation. So far, therapeutic options for ALS are extremely minimal and ineffective therefore, multi-faceted approaches such as treating the oxidative stress and inhibiting the TDP-43s aggregation are being actively pursued. In our recent study, an acridine imidazolium derivative compound, AIM4, has been identified to have anti-TDP-43 aggregation propensity however, its mechanism of inhibition is not deciphered. In this study, we have utilized computational methods to examine binding site(s) of AIM4 in the TDP-43 structure and have also compared its binding efficiency with several other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic core region of amino acids aa: 288-319, which coincides with one of the key residue motifs that could potentially mediate liquid-liquid phase separation (LLPS) of TDP-43. Importantly, alike to the previously reported effects exerted by RNA molecules, we found that AIM4 could also inhibit the in vitro LLPS of a recombinantly purified C-terminal fragment TDP-432C bearing an A315T familial mutation. Antagonistic effects of AIM4 towards LLPS which is believed as the precursor process to the TDP-43s aggregation and the in silico prediction of a binding site of AIM4 on TDP-43 occurring in the same region, assert that AIM4 could be an important molecule for further investigations on TDP-43s anti-aggregation effects with relevance to the ALS pathogenesis.

bioinformatics↗

bioRxiv2018

Detection of residual disease after neoadjuvant therapy in breast cancer using personalized circulating tumor DNA analysis

Accurate detection of minimal residual disease (MRD) can guide individualized management of early stage cancer patients, but current diagnostic approaches lack adequate sensitivity. Circulating tumor DNA (ctDNA) analysis has shown promise for recurrence monitoring but MRD detection immediately after neoadjuvant therapy or surgical resection has remained challenging. We have developed TARgeted DIgital Sequencing (TARDIS) to simultaneously analyze multiple patient-specific cancer mutations in plasma and improve sensitivity for minute quantities of residual tumor DNA. In 77 reference samples at 0.03%-1% mutant allele fraction (AF), we observed 93.5% sensitivity. Using TARDIS, we analyzed ctDNA in 34 samples from 13 patients with stage II/III breast cancer treated with neoadjuvant therapy. Prior to treatment, we detected ctDNA in 12/12 patients at 0.002%-1.04% AF (0.040% median). After completion of neoadjuvant therapy, we detected ctDNA in 7/8 patients with residual disease observed at surgery and in 1/5 patients with pathological complete response (odds ratio, 18.5, Fishers exact p=0.032). These results demonstrate high accuracy for a personalized blood test to detect residual disease after neoadjuvant therapy. With additional clinical validation, TARDIS could identify patients with molecular complete response after neoadjuvant therapy who may be candidates for nonoperative management.\n\nOne Sentence SummaryA personalized ctDNA test achieves high accuracy for residual disease.

genomics↗