Biology subjects

Bhatia, P.

Publications and source records attributed to Bhatia, P..

4 recordsLinked to original sources

bioRxiv2020

Critical role for cold shock protein YB-1 in cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and cancer progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cell lines resulted in cytokinesis failure, multinucleation and an increase in G1 transit time. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging of cells undergoing cytokinesis both in vitro and in zebrafish embryos, we found that YB-1 was critical for microtubule organization during cytokinesis. Using mass spectrometry we identified multiple novel phosphorylation sites on YB-1. We show that phosphorylation of YB-1 at multiple serine residues was essential for its function during cytokinesis. Using atomistic modelling we show how multiple phosphorylations alter YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining for the first time precisely how YB-1 regulates cell division. SUMMARYY-box-binding protein-1, YB-1, is essential for cell division, but it is not clear how it functions. Using live imaging and confocal microscopy we show that YB-1 functions only in the last step of division, specifically being required to initiate cytokinesis.

cell biology↗

bioRxiv2019

Intelli-NGS: Intelligent NGS, a deep neural network-based artificial intelligence to delineate good and bad variant calls from IonTorrent sequencer data

BackgroundIonTorrent is a second-generation sequencing platform with smaller capital costs than Illumina but is also prone to higher machine error than later. Given its lower costs, the platform is generally preferred in developing countries where next-generation sequencing is still a very exclusive technique. There are many software tools available for other platforms but IonTorrent. This makes the already tricky analysis part more error-prone. MotivationWe have been using the IonTorrent platform in our hospital setting for aiding diagnosis or treatment for the past couple of years. Given to our experience, analysis part of IonTorrent data takes the longest time and still, we used to get stuck with certain variants which seemed fine on looking at their metrics but were found to be negative in Sanger sequencing verification. This made us determined to develop a tool that could aid us in reducing false positive and negative rates while still retaining good recall. The artificial intelligence-based technique was our final choice after developing pipelines with less success. MethodologyThe artificial intelligence was developed from scratch in Python 3 using TensorFlow fully connected dense layers. The model takes VCF files as input and solves each variant based on the thirty-five parameters given by the IonTorrent platform, including the flow-space information which is missed by variant callers other than the default torrent variant caller. ResultsThe final trained model was able to achieve an accuracy of 93.08% and a ROC-AUC of 0.95 with GIAB validation data. The additional program that was written to run the model annotates each variant using online databases such as dbSNP, ClinVar and others. A probability score for each outcome for each variant is also provided to aid in decision making. AvailabilityThe model and running code are available for free only for non-commercial users at https://www.github.com/aditya-88/intelli-ngs.

bioinformatics↗

bioRxiv2019

Effective downregulation of BCR-ABL tumorigenicity by RNA targeted CRISPR-Cas13a

AimTo induce BCR-ABL gene silencing using CRISPR Cas13a. BackgroundCML is a clonal myeloproliferative disorder of pluripotent stem cells driven by a reciprocal translocation between chromosome 9 and 22, forming a BCR-ABL fusion gene. Tyrosinekinase inhibitor drugs like imatinib are the mainstay of treatment and cases resistant to these drugs have a poor prognosis in the absence of a compatible stem-cell donor. However, with rapid advancements in gene-editing technologies, most studies are now focusing on developing a translational model targeting single-gene disorders with a prospective permanent cure. ObjectiveTo explore the potential application of the RNA targeting CRISPR-Cas13a system for effective knockdown of BCR-ABL fusion transcript in a CML cell line, K562. MethodCRISPR Cas13a crRNA was designed specific to the chimeric BCR-ABL gene and the system was transfected as a two-plasmid system into a CML cell line, K562. The effects were enumerated by evaluating the expression levels of downstream genes dependent on the expression of the BCR-ABL gene. Also, next-generation sequencing was used to ascertain the effects of CRISPR on the gene. ResultsThe CRISPR system was successfully able to lower the expression of downstream genes (pCRKL and pCRK) dependent on the activated BCR-ABL kinase signal by up-to 4.3 folds. The viability of the CRISPR treated cells were also significantly lowered by 373.83-fold (p-value= 0.000891196). The time-dependent kinetics also highlighted the significant in-vitro suppressive activity to last up to 8 weeks (p-value: 0.025). As per the cDNA sequencing data from Oxford MinION next-generation sequencer, the CRISPR treated cells show 62.37% suspected cleaved reads. ConclusionThese preliminary results highlight an excellent potential application of RNA targeting CRISPRs in Haematological neoplasms like CML and should pave way for further research in this direction.

molecular biology↗

bioRxiv2019

Massive parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a novel genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction is quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine variants effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino-acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in 2,398 patients treated with thiopurines, with 100% sensitivity and specificity, in contrast with poor performance of bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,103 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.

genomics↗