Biology subjects

Parvez, S.

Publications and source records attributed to Parvez, S..

8 recordsLinked to original sources

bioRxiv2026

A dCas9 Proximity Reporter for Investigating DNA Linear and Rotational Dynamics

The dCas9 system has rapidly been developed into many tools to explore different aspects of the human genome; the high binding specificity, coupled with the inactive nuclease enzyme, allows for precise recruitment of molecules to specific sequences of DNA. We sought to exploit these capabilities to create a tool for assessing the real-time proximity of two DNA sequences within a biological system. By incorporating aptamers into gRNAs, dCas9 molecules can be used to recruit the {beta}9 or {beta}10 strands of split-NanoLuc(R) to specific DNA sequences and quantify the proximity of those sequences based on their ability to complex with the luciferase fragment ({Delta}11S) and produce luminescence. While many tools exist to detect a single DNA sequence, this system is uniquely capable of assessing how two DNA sequences interact with each other. As expected, we found that the interaction of two dCas9 molecules was affected by their linear distance from each other on dsDNA. Surprisingly, we found that their interaction was also strongly influenced by rotational orientation, even for sequences that are close together in linear space. This finding indicates that dCas9 rotational alignment is an important consideration for designing dCas9 systems that target multiple DNA sequences simultaneously. Beyond the findings presented herein, we believe this DNA proximity detection tool has the potential to be adapted for applications involving the proximity and orientation of two DNA sequences.

bioengineering↗

bioRxiv2025

Spike protein E2 of chikungunya virus: a plant-based vaccine exhibited potent immunogenicity in BALB/c mice

Over the last two decades, chikungunya virus (CHIKV) infections have surged worldwide, causing significant suffering. CHIKV E2 gene codes for spike protein E2, essential for virus-host interactions and thus serves as a potential vaccine candidate. We expressed a full-length E2 (S27 African prototype) in both E. coli and Nicotiana tabacum, which triggered an immune response in BALB/c mice. First, E2 was computationally analyzed for PTM patterns and prediction of B-cell and T-cell epitopes. Next, molecular docking of epitopes with MHCs (Class I and II) revealed high affinity, confirmed by Molecular Dynamics Simulation. Then, the chemically synthesized E2-6xHis tag was cloned and expressed in both E. coli and N. tabacum under T7 and CaMV promoters, respectively. E2 was cloned into the pUC57 vector (E. coli) and expressed using the pET28a(+) vector in BL21(DE3)pLysS cells, followed by cloning into the pCAMBIA1302 vector and transformation into Agrobacterium tumefaciens. RT-PCR and confocal visualization confirmed the formation of E2 transcripts. Recombinant E2 was purified on Ni-NTA columns and visualized as a protein of [~]49 kDa on SDS PAGE. Finally, E2 was injected into BALB/c mice; neutralizing antibodies, including IgG, were detected as positive in the indirect ELISA, with the highest levels observed at 3 days post-infiltration (3 dpi). Western blot also confirmed E2 expression in E coli and tobacco, and induction of E2-specific antibodies in BALB/c mice. This study presents a promising approach to developing a safe and effective vaccine against chikungunya fever in plants.

molecular biology↗

bioRxiv2025

MIC-Drop-seq: Scalable single-cell phenotyping of mutant vertebrate embryos

Advances in genome engineering and single-cell RNA sequencing (scRNAseq) have revolutionized the ability to precisely map gene functions, yet scaling these techniques for large-scale genetic screens in animals remains challenging. We combined high-throughput gene disruption in zebrafish embryos via Multiplexed Intermixed CRISPR Droplets with phenotyping by multiplexed scRNAseq (MIC-Drop-seq). In one MIC-Drop-seq experiment, we intermixed and injected droplets targeting 50 transcriptional regulators into 1,000 zebrafish embryos, followed by pooled scRNAseq. Tissue-specific gene expression and cell abundance analysis of demultiplexed mutant cells recapitulated many known phenotypes, while also uncovering novel functions in brain and mesoderm development. We observed pervasive cell-extrinsic effects among these phenotypes, highlighting how whole-embryo sequencing captures complex developmental interactions. Thus, MIC-Drop-seq provides a powerful and scalable platform for mapping gene functions in vertebrate development with cellular resolution.

genomics↗

bioRxiv2022

Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase

The zebrafish Danio rerio has become a popular model in functional genomics and genetic disease studies. However, when a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, using standard genotyping methods to identify a population of homozygous mutant embryos is time-consuming and sometimes impractical due to downstream applications such as large-scale chemical screens. Here, we introduce TICIT, Targeted Integration by CRISPR-Cas9 and Integrase Technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert fluorescent markers into CRISPR-Cas9-generated mutant alleles. It allows instantaneous determination of a zebrafishs genotype simply by examining its color. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to insert large DNA fragments at the same genomic location repeatedly and with high precision and efficiency. We demonstrated that TICIT could also be used to create reporter fish driven by an endogenous promoter. Additionally, we created a landing site located in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types, acting as a putative safe harbor locus. Hence, TICIT can yield predictable and reproducible transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms.

bioengineering↗

bioRxiv2022

Z-REX uncovers a bifurcation in function of Keap1 paralogs

Studying electrophile signaling is marred by difficulties in parsing changes in pathway flux attributable to on-target, vis-a-vis off-target, modifications. By combining bolus dosing, knockdown, and Z-REX--a tool investigating on-target/on-pathway electrophile signaling, we document that electrophile labeling of one zebrafish-Keap1-paralog (zKeap1b) stimulates Nrf2-driven antioxidant response (AR) signaling (like the human-ortholog). Conversely, zKeap1a is a dominant-negative regulator of electrophile-promoted Nrf2-signaling, and itself is nonpermissive for electrophile-induced Nrf2-upregulation. This behavior is recapitulated in human cells, wherein following electrophile treatment: (1) zKeap1b-transfected cells are permissive for augmented AR-signaling through reduced zKeap1b-Nrf2 binding; (2) zKeap1a-transfected cells are non-permissive for AR-upregulation, as zKeap1a-Nrf2 binding capacity remains unaltered; (3) 1:1 ZKeap1a:zKeap1b-transfected cells show no Nrf2-release from the Keap1-complex, rendering these cells unable to upregulate AR. We identified a zKeap1a-specific point-mutation (C273I) responsible for zKeap1as behavior. Human-Keap1(C273I), of known diminished Nrf2-regulatory capacity, dominantly muted electrophile-induced Nrf2-signaling. These studies highlight divergent and interdependent electrophile signaling behaviors, despite conserved electrophile sensing. Impact statementHow electrophile-sensing versus -signaling marshal stress responses: two zebrafish Keap1-paralogs are equally adept at electrophile-sensing but manifest divergent and co-regulatory electrophile-signaling behaviors.

pharmacology and toxicology↗

bioRxiv2022

Z-REX: Shepherding Reactive Electrophiles to Specific Proteins Expressed either Tissue-Specifically or Ubiquitously, and Recording the Resultant Functional Electrophile-Induced Redox Responses in Larval Fish

Summary of the Protocol ExtensionThis Protocol Extension describes the adaptation of an existing Nature Protocol detailing the use of T-REX (targetable reactive electrophiles and oxidants)--an on-demand redox targeting toolset in cultured cells. The adaptation described here is for use of REX technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)--either ubiquitously or tissue-specifically--are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments). The reactive electrophile is then photouncaged at a user-defined time, enabling proximity-assisted electrophile-modification of a POI. Functional and phenotypic ramifications of POI-specific modification can then be monitored, by coupling to standard downstream assays, such as, Click chemistry-based POI-labeling and target-occupancy quantification; immunofluorescence or live imaging; RNA-Seq and qRT-PCR analyses of downstream-transcript modulations. Transient expression of requisite Halo-POI in zebrafish embryos is achieved by mRNA injection. Procedures associated with generation of transgenic zebrafish expressing a tissue-specific Halo-POI are also described. The Z-REX experiments can be completed in <1-week using standard techniques. To successfully execute Z-REX, researchers should have basic skills in fish husbandry, imaging, and pathway analysis. Experience with protein or proteome manipulation is useful. This protocol extension is aimed at helping chemical biologists study precision redox events in a model organism and fish biologists perform redox chemical biology.

biochemistry↗

bioRxiv2020

Deletion of Autophagy gene ATG1 and F-box motif encoding gene YDR131C together leads to Synthetic Growth Defects and Flocculation behaviour in Saccharomyces cerevisiae

F-box motif encoding YDR131C is functionally uncharacterized gene which forms the complex with the SCF-E3 ligase. The F-box motif containing proteins are involved in substrate recruitment for the ubiquitination and subsequent degradation through 26S proteasome. Autophagy gene, ATG1 (ULK1in human) is a well conserved serine-threonine kinase, required for vesicle formation and cytoplasm to vacuole targeting pathway. Atg1p forms the complex with Atg13p and Atg17p during autophagy. The understanding of crosstalk between ubiquitin and autophagy pathways is crucial for synthetic lethality screen and drug targeting. Here we have conducted the study for genetic interaction between uncharacterized YDR131C and ATG1 gene representing both specific and non-specific protein degradation pathways. The single and double gene knockout strains of YDR131Cand ATG1 genes were constructed in the BY4741 genetic background and analysed for growth fitness. The strains were also evaluated for cellular growth response in presence of hydroxyurea (HU), methyl methane sulfonate (MMS), and hydrogen peroxide (H2O2) stress causing agents by spot assay. The ydr131c{Delta}atg1{Delta} showed the synthetic growth defect phenotype with floc formation in rich medium which showed floc disruption in presence of EDTA. The ydr131c{Delta}atg1{Delta} cells showed the sensitivity to stress agents HU, MMS, and H2O2 when compared with ydr131c{Delta}, atg1{Delta}, and WT cells.. Based on the observations, we report that YDR131C and ATG1 functions in parallel pathways for growth fitness and cellular growth response to stress agents. Interestingly this study also revealed the crosstalk between ubiquitination and autophagy pathways. The defects in both the pathways could lead to synthetic growth defects which may have implication for the precision medicine initiatives.

genetics↗

bioRxiv2020

Deletion of Autophagy gene ATG1 and Glyoxylate cycle Regulator encoding gene UCC1 together leads to Synthetic Growth Defects and Sensitivity to Genotoxic agents in Saccharomyces cerevisiae

Atg1 of S. cerevisiae is a key component of autophagy encoded by ATG1 gene, involved in the process of degradation of cytosolic components through autophagy. UCC1, an F-box encoding gene is involved in the negative regulation of glyoxylate pathway via degradation of Cit2 enzyme by ubiquitin proteasome system. We investigated the genetic interaction between ATG1 and UCC1 using the gene deletion approach. The atg1{Delta}ucc1{Delta} cells showed the synthetic growth defects with abnormal budding and sensitivity to genotoxic and oxidative stress agents. Based on the observations, we report that ATG1 and UCC1 interact genetically to regulate the cell growth fitness and function in parallel pathway in cellular response to the genotoxic stress agents. The present investigation also revealed the cross talks among autophagy, ubiquitin proteasome system, and glyoxylate pathways.

genetics↗