Biology subjects

Bhatia, R.

Publications and source records attributed to Bhatia, R..

7 recordsLinked to original sources

bioRxiv2026

Deep Brain Stimulation Microelectrodes as a Source of Human Subcortical RNA: Validation of a Low-Input Transcriptomic Protocol

BackgroundUnderstanding the molecular basis of Parkinsons disease (PD) phenotypic heterogeneity maybe improved by in vivo access to deep brain tissue. Deep brain stimulation (DBS) surgery offers a unique opportunity: microelectrodes traversing the subthalamic nucleus (STN) carry adherent brain tissue upon withdrawal, providing a source of RNA from subcortical regions in living patients without additional invasive procedures. AimTo develop and validate a low-input RNA extraction and transcriptomic profiling protocol using DBS microelectrodes in post-mortem human brain, to confirm that recovered RNA is of brain rather than blood origin, and to characterise its sub-regional and cellular identity. MethodologyDBS microelectrodes were inserted without image guidance or guide tube into three unfixed post-mortem human brains targeting the STN trajectory. In clinical practice, a guide tube shields the electrode from cortical tissue; its absence here means tissue from the full insertion trajectory may contribute to recovered RNA. Thirty-eight microelectrodes were evaluated across single and pooled strategies. RNA was extracted using a modified RNeasy Micro low-input protocol; libraries prepared using NEBNext Single Cell/Low Input RNA Library Kit and sequenced on Illumina NovaSeq 6000 (paired-end, 2x150 bp). Tissue identity was validated against GTEx v10 (54 tissues) and Allen Human Brain Atlas (ABA, 19 subcortical regions including STN) using Pearson correlation with permutation testing (1,000 permutations) and BH-FDR correction. Transcriptional overlap between subcortical and cortical reference regions was quantified and subcortical-enriched gene filtering performed. ResultsTwenty-five RNA isolates were obtained from 38 microelectrodes; 54.5% of Bioanalyzer-assessed samples achieved RIN [≥]5 (median 7.1; range: 5.9-7.8). Fifteen libraries passed sequencing QC (mean depth 43.0 {+/-} 14.1 million read pairs; mean Q30 83.2 {+/-} 5.6 %). PCA of rlog transformed expression data resolved samples by donor identity. All samples confirmed brain tissue origin by GTEx {tau}-index tissue specificity correlation (n=996 brain and blood specific markers). Brain correlations were highest for frontal cortex (mean r= 0.683 {+/-} 0.072), anterior cingulate cortex (mean r= 0.658 {+/-} 0.069), amygdala (mean r= 0.615 {+/-} 0.068), and basal ganglia including caudate (mean r= 0.553 {+/-} 0.059), putamen (mean r= 0.546 {+/-} 0.059), and nucleus accumbens (mean r= 0.544 {+/-} 0.057); all BH-FDR < 0.05. Cerebellum showed the lowest brain correlations (cerebellar hemisphere: mean r = 0.327 {+/-} 0.039; cerebellum: mean r = 0.314 {+/-} 0.037). Whole blood correlation was strongly negative (mean r= -0.250 {+/-} 0.079), confirming non-haematological origin. ABA genome-wide analysis confirmed positive STN correlation (r=0.54-0.62; permutation p<0.001). MuSiC cell-type deconvolution against the Allen Brain Atlas HMBA-BG snRNA-seq reference identified oligodendrocytes (33.5 {+/-} 17.2%), frontal cortical neurons (9.1 {+/-} 7.0%), STR D1 MSNs (6.0 {+/-} 4.9%), and dopaminergic neurons (1.1{+/-} 2.7%) as the principal cell types, independently corroborating bulk transcriptomic findings at single-cell resolution. ConclusionThis study validates a low-input RNA extraction protocol for DBS microelectrodes, confirming brain-specific transcriptomic profiles consistent with STN-adjacent subcortical sampling. The 96% transcriptional overlap between cortical and subcortical regions, combined with absence of a guide tube in this post-mortem model, limits sub-regional specificity; clinical application with a guide tube would enrich the subcortical signal. These findings provide the methodological framework for in vivo molecular profiling of the human basal ganglia during DBS surgery in PD patients.

genetics↗

bioRxiv2025

Unlocking the Power of CXCR2 Inhibition to Overcome Gemcitabine Resistance in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality, characterized by intrinsic resistance to conventional therapies and limited effective treatment options. In this study, we investigated the role of the CXCR2 axis in PDAC therapy resistance. CXCR2, a chemokine receptor, is actively involved in inflammation, tumor angiogenesis, and metastasis. Our working hypothesis is that CXCR2 contributes to PDAC chemotherapy resistance. To test this, we generated gemcitabine-resistant (GemR) lines using T3M4 and CD18/HPAF (CD18) cell lines. Baseline expression of CXCL1, CXCL5, and CXCL8 ligands were higher in GemR cells compared to parental cells. Upon gemcitabine treatment, parental cells exhibited a greater increase in CXCL1 and CXCL8 expression than GemR cells. Further analysis in T3M4 cells revealed a dose- and time-dependent increase in CXCL1 and CXCL8 expression following gemcitabine exposure. Next, we assessed whether targeting CXCR2 could enhance the therapeutic response. We treated parental and GemR cell lines with gemcitabine in combination with the CXCR2 antagonist SCH 479833 (SCH 47). Notably, lower concentrations of gemcitabine combined with SCH 47 were more effective than higher concentrations of gemcitabine alone in GemR cell lines. In both parental and GemR xenograft models, combination therapy with Navarixin (a CXCR2 antagonist) and gemcitabine demonstrated superior antitumor and antimetastatic activity compared to either treatment alone. In conclusion, these findings highlight the critical role of the CXCR2 axis in PDAC therapy resistance. Targeting CXCR2 enhances gemcitabine efficacy, offering a potential therapeutic strategy to overcome resistance in PDAC.

cancer biology↗

bioRxiv2022

Pan-cancer Analysis Predicts Kindlin-associated Global Mechanochemical Perturbation

Kindlins are mechanosensitive adapter proteins that connect extracellular mechanical cues to intracellular chemical events. Any alterations in these proteins thus alter cellular signaling, which could result in cancer progression. However, their involvement in global mechanochemical signals remains elusive in cancers. Here we analyze pan-cancer samples to decipher how kindlin alterations aid cancer progression. We show that kindlin alterations, at both the genetic and mRNA level, dysregulates cellular behavior which significantly correlate with poor survival. We find that while these alterations are cancer-specific, they are prevalent in advanced tumor stages and metastatic onset. We observe that kindlins co-alter with a substantial fraction of human mechanochemical proteome in various tumors. Our analysis suggests how kindlin alterations aid tumor-promoting signals with a synergistic effect from alterations of cancer-hallmark genes. Notably, we demonstrate a consistent alteration of epithelial-mesenchymal-transition markers with kindlin activity. Overall, our study highlights how kindlin alterations could affect metabolism, genomic instability, and signal disruption via their interactome network, causing cancer and suggests targeting them as a therapeutic strategy.

cancer biology↗

bioRxiv2021

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time

Ribosomal subunit biogenesis within mammalian cells initiates in the nucleolus with the assembly of a 90S precursor particle, which is subsequently split into the pre-40S and pre-60S subunits. During further processing steps, pre-ribosomal subunits are loaded with export receptors, which enables their passage through the pore complexes (NPCs) into the cytoplasm. Here export factors are released and both subunits can form a mature ribosome. Ribosomal biogenesis has been studied in great detail by biochemical, genetic and electron microscopic approaches, however, until now live cell data on the in vivo kinetics are still missing. We analysed export kinetics of the large ribosomal subunit ("pre-60S particle") through single NPCs in living human cells. To assess the in vivo dynamics of this process, we established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label ribosomal 60S subunits (eIF6) and NPCs (NTF2). By combining single molecule tracking and super resolution confocal microscopy in a highly customized microscopic setup, we visualized the dynamics of single pre-60S particles during the interaction with and export through single NPCs. In this way we obtained unprecedented insights into this key cellular process. Our results revealed that for export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export process takes place with a single rate limiting step and an export dwell time of ~24 milliseconds. Only about 1/3 of attempted export events were successful. Given the molecular mass of the pre-60S particles our results show that the mass flux through a single NPC can reach up to ~125 MDa{middle dot}s-1 in vivo.

cell biology↗

bioRxiv2020

Methylation of Dual Specificity Phosphatase 4 Controls Cell Differentiation

A collection of signaling and epigenetic events needs to be orchestrated for normal development of hematopoietic lineages. While mitogen-activated protein (MAP) kinases (MAPKs) and multiple epigenetic modulators have been implicated in the megakaryocytic (Mk) cell differentiation, the underlying molecular mechanisms of signaling-epigenetic crosstalk remain unclear. MAPKs are in general inactivated by dual specificity phosphatases (DUSPs), whose activities are tightly regulated by various posttranslational modifications. Using knockdown screening and single-cell transcriptional analysis, we determined that DUSP4 is the phosphatase that inactivates p38 MAPK in hematopoietic cells and serves as a key regulator to promote Mk differentiation. With the nextgeneration Bioorthogonal Profiling of Protein Methylation technology for live cells, we identified DUSP4 as a PRMT1 substrate. Mechanistically, PRMT1-mediated Arg351 methylation of DUSP4 triggers its ubiquitinylation by HUWE1 (an E3 ligase) and then degradation, which results in p38 MAPK activation and inhibition of Mk differentiation in vitro and in vivo. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation and thus argues immunological roles of Mk cells. Collectively, these results demonstrate a critical role of PRMT1-mediated posttranslational modification of DUSP4 in regulation of Mk differentiation and maturation. In the context of thrombocytopenia observed in myelodysplastic syndromes (MDS), we demonstrated that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis in MDS samples. These findings provide novel mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a targeting strategy for treatment of thrombocytopenia associated with myeloid malignancies such as MDS.

cell biology↗

bioRxiv2020

GSK3 inhibition rescues growth and telomere dysfunction in dyskeratosis congenita iPSC-derived type II alveolar epithelial cells

Dyskeratosis congenita (DC) is a rare genetic disorder characterized by deficiencies in telomere maintenance leading to very short telomeres and the premature onset of certain age-related diseases, including pulmonary fibrosis (PF). PF is thought to derive from epithelial failure, particularly that of type II alveolar epithelial (AT2) cells, which are highly dependent on Wnt signaling during development and adult regeneration. We use human iPSC-derived AT2 (iAT2) cells to model how short telomeres affect AT2 cells. Cultured DC mutant iAT2 cells accumulate shortened, uncapped telomeres and manifest defects in the growth of alveolospheres, hallmarks of senescence, and apparent defects in Wnt signaling. The GSK3 inhibitor, CHIR99021, which mimics the output of canonical Wnt signaling, enhances telomerase activity and rescues the defects. These findings support further investigation of Wnt agonists as potential therapies for DC related pathologies.

cell biology↗

bioRxiv2019

Doubled haploid based parental lines are most suitable in predicting heterosis using microsatellites and in development of highly heterotic F1 hybrids in Brassica oleracea

In Brassica oleracea, heterosis is one of the most efficient tools giving impetus to hybrid vegetable industry. In this context, we presented the first report on identifying superior heterotic crosses for yield and commercial traits in cauliflower involving cytoplasmic male sterile (CMS) and doubled haploid (DH) lines as parents. We studied the suitability of SSR and EST-SSRs based genetic distance (GD) and morphological markers based phenotypic distance (PD) in prediction of heterosis when DH based genotypes are used as parents in developing F1 hybrids. Overall 120 F1 hybrids derived from twenty Ogura cybrid CMS lines and six DH based testers were evaluated for 16 phenotypic traits along with their 26 parental lines and 4 commercial standard checks, in 10 x 15 alpha lattice design. The genomic SSR and EST-SSRs based genetic structure analysis grouped 26 parental lines into 4 distinct clusters. The CMS lines Ogu118-6A, Ogu33A, Ogu34-1A were good general combiner for developing short duration hybrids. The SCA effects were significantly associated with heterosis suggesting non-additive gene effects for heterotic response of hybrids. Less than unity value of {sigma}2A/D coupled with {sigma}2gca/{sigma}2scaindicated the predominance of non-additive gene action in the expression of studied traits. The genetic distance estimates among 26 parents ranged from 0.44 to 0.98 and were significantly associated with heterosis for important commercial traits, suggesting the utility of microsatellite based genetic distance in prediction of heterosis in B. oleracea.

plant biology↗