Biology subjects

Yadav, N. S.

Publications and source records attributed to Yadav, N. S..

8 recordsLinked to original sources

bioRxiv2026

Encapsulation in a bacterial microcompartment shell improves thermal stability of a glycolytic enzyme

Selective encapsulation of target enzymes is an increasingly well studied field with a host of potential applications for biotechnology. Natively, many bacteria utilize bacterial microcompartments (BMCs) for enzyme encapsulation to enhance catalysis. BMCs are protein shells that enable selective localization of targeted metabolic enzymes and may improve catalytic rates by co-localizing pathway enzymes and/or serve to sequester toxic or volatile intermediates. The microcompartment shell of Haliangium ochraceum (HO) is a notable BMC chassis because of its modularity and versatility; it is easily expressed and assembled outside its native host and can accept a wide array of cargo. Recently, it was demonstrated that assembly of HO BMC shells can be easily achieved in vitro. Following up on our previous work on in vivo assembly of HO-BMCs with triose phosphate isomerase (TPI) as model enzyme cargo, here we have demonstrated the advantages of in vitro assembly (IVA) for targeted enzyme encapsulation. We achieved variable loading of BMC shells with targeted amounts of TPI and demonstrated enhanced thermal stability of encapsulated TPI versus free TPI up to 62{degrees}C.

synthetic biology↗

bioRxiv2024

Structure Characterization of Bacterial Microcompartment Shells via X-ray Scattering and Coordinate Modeling: Evidence for adventitious capture of cytoplasmic proteins

Bacterial microcompartments (BMCs) are self-assembling, protein shell structures that are widely investigated across a broad range of biological and abiotic chemistry applications. A central challenge in BMC research is the targeted capture of enzymes during shell assembly. While crystallography and cryo-EM techniques have been successful in determining BMC shell structures, there has been only limited success in visualizing the location of BMC-captured enzyme cargo. Here, we demonstrate the opportunity to use small angle X-ray scattering (SAXS) and pair density distribution function (PDDF) measurements combined with quantitative comparison to coordinate structure models as an approach to characterize BMC shell structures in solution conditions directly relevant to biochemical function. Using this approach, we analyzed BMC shells from Haliangium ochraceum that were isolated following expression in E. coli. The analysis allowed BMC shell structures and the extent of encapsulated enzyme cargo to be identified. Notably, the results demonstrate that HO-BMC shells adventitiously capture significant amounts of cytoplasmic cargo during assembly in E. coli. Our findings highlight the utility of SAXS/PDDF analysis for evaluating BMC architectures and enzyme encapsulation, offering valuable insights for designing BMC shells as platforms for biological and abiotic catalyst capture within confined environments.

biophysics↗

bioRxiv2021

Gibberellins target shoot-root growth, morpho-physiological and molecular pathways to induce cadmium tolerance in mung bean

Cadmium (Cd) inhibits plant growth, perturb nutrient uptake and affect chloroplast ultra structure. Cd soil pollution is mainly contributed by excessive use of phosphate fertilizers, nickel Cd batteries, plating and sewage sludge. Research investigations deciphering role of Cd in affecting overall performance of mung bean is least understood. Likewise ameliorative effects of gibberellins (GAs) in Cd induced toxicity in mung bean are lesser known. In this context, effects of Cd stress (CdCl2, IC50 -500 {micro}M L-1) with or without GA3 application on mung bean (Vigna radiata L. Var. SML-668) plants were comprehensively investigated under controlled conditions. In brief, a total of 80 mung bean plants (15 days old of uniform height) were divided into four groups, with each group (n=20) subjected to four different treatments (Control, CdCl2, GA3, CdCl2+GA3), twice during the entire life cycle of mung bean plants (until harvest 85-90 days). Results revealed negative impacts of Cd stress on shoot morphometry (plant height, leaf surface area, stem diameter, shoot fresh weight, number of leaves, number of pods, length and diameter of pods), root morphometry (root length, root surface area, root dry weight, nodule number and nodule diameter), photosynthetic pigments and agronomic traits. GA3 ameliorated Cd stress by modulating shoot and root growth rates, improving overall plant metabolism, photosynthetic pigments, and shoot and root morphometry and transcript abundance of VgPCS1, VgPCS2, VgCdR and VgIRT1. Current study proposes GA3 application for the effective management of Cd induced phytotoxicity in mung bean plants.

plant biology↗

bioRxiv2020

Multigenerational exposure to heat stress induces phenotypic resilience, and genetic and epigenetic variations in Arabidopsis thaliana offspring

Plants are sedentary organisms that constantly sense changes in their environment and react to various environmental cues. On a short-time scale, plants respond through alterations in their physiology, and on a long-time scale, plants alter their development and pass on the memory of stress to the progeny. The latter is controlled genetically and epigenetically and allows the progeny to be primed for future stress encounters, thus increasing the likelihood of survival. The current study intended to explore the effects of multigenerational heat stress in Arabidopsis thaliana. 25 generations of Arabidopsis thaliana were propagated in the presence of heat stress. The multigenerational stressed lineage F25H exhibited a higher tolerance to heat stress and elevated frequency of homologous recombination, as compared to the parallel control progeny F25C. A comparison of genomic sequences revealed that the F25H lineage had a three-fold higher number of mutations (SNPs and INDELs) as compared control lineages, suggesting that heat stress induced genetic variations in the heat-stressed progeny. The F25H stressed progeny showed a 7-fold higher number of non-synonymous mutations than the F25C line. Methylome analysis revealed that the F25H stressed progeny showed a lower global methylation level in the CHH context than the control progeny. The F25H and F25C lineages were different from the parental control lineage F2C by 66,491 and 80,464 differentially methylated positions (DMPs), respectively. F25H stressed progeny displayed higher frequency of methylation changes in the gene body and lower in the body of transposable elements (TEs). Gene Ontology analysis revealed that CG-DMRs were enriched in processes such as response to abiotic and biotic stimulus, cell organizations and biogenesis, and DNA or RNA metabolism. Hierarchical clustering of these epimutations separated the heat stressed and control parental progenies into distinct groups which revealed the non-random nature of epimutations. We observed an overall higher number of epigenetic variations than genetic variations in all comparison groups, indicating that epigenetic variations are more prevalent than genetic variations. The largest difference in epigenetic and genetic variations was observed between control plants comparison (F25C vs F2C), which clearly indicated that the spontaneous nature of epigenetic variations and heat-inducible nature of genetic variations. Overall, our study showed that progenies derived from multigenerational heat stress displayed a notable adaption in context of phenotypic, genotypic and epigenotypic resilience.

plant biology↗

bioRxiv2020

Development and optimization of a germination assay and long-term storage for Cannabis sativa pollen

Pollen viability and storage is of great interest to cannabis breeders and researchers to maintain desirable germplasm for future use in breeding or for biotechnological and gene editing applications. Here, we report a simple and efficient cryopreservation method for long-term storage of Cannabis sativa pollen. Additionally, we have deciphered the bicellular nature of cannabis pollen using DAPI staining. We have also standardized a pollen germination assay to assess the viability of cannabis pollen, and found pollen collected from different principal growth stages exhibits different longevity. Finally, we developed a long-term storage method which includes pollen combination with baked whole wheat flower and desiccation under vacuum for cryopreservation. By using this method, we were able to maintain germination viability in liquid nitrogen after 4 months, suggesting potentially indefinite preservation of cannabis pollen.

plant biology↗

bioRxiv2020

Development of transient expression assay for Cannabis sativa which revealed differential Agrobacterium susceptibility among cannabis cultivars

In plant biology, transient expression analysis plays a vital role to provide a fast method to study the gene of interest and subsequently leads the path to develop an improved crop variety with better agronomic traits. In this study, we have reported a rapid and efficient method for transient expression in Cannabis sativa seedlings using Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 carrying the pCAMBIA1301 construct with uidA gene was used to transform cannabis seedlings and the GUS assay was used to detect the uidA expression. A 1% hydrogen peroxide (H2O2) solution was used for both seed sterilization and rapid germination steps. Transient transformation revealed that both cotyledons and young true leaves are amenable to transformation. Comparison to Nicotiana tabacum (tobacco) showed that cannabis seedlings were less susceptible to transformation with Agrobacterium tumefaciens. The susceptibility to Agrobacterium infection also varied with the different cannabis cultivars. The method established in this study has potential to be an important tool for gene-function studies and genetic improvement in cannabis.

molecular biology↗

bioRxiv2019

Computational and experimental evidence to the permeability of withanolides across the normal cell membrane

Poor bioavailability due to the inability to cross the cell membrane is one of the major reasons for the failure of a drug in the clinical trials. We have used molecular dynamics simulations to predict the membrane permeability of natural drugs - withanolides (withaferin-A and withanone) that have similar structures but remarkably differ in their cytotoxicity. We found that withaferin-A, but not withanone, could proficiently transverse through the model membrane. The free energy profiles obtained were in accordance with the physico-chemical properties of the investigated drug molecules. It was observed that the polar head group of the bilayer exhibits high resistance for the passage of withanone as compared to withaferin-A, while the interior of the membrane behaves similarly for both withanolides. The solvation analysis revealed that the high solvation of terminal O5 oxygen of withaferin-A was the major driving force. The impact of the favorable interaction of terminal oxygen (O5) of withaferin-A with the phosphate of the membrane led to its smooth passage across the bilayer. The computational predictions were validated by raising and recruiting unique antibodies that react to withaferin-A and withanone. Further, the time-lapsed analyses of control and treated human normal and cancer cells, demonstrated proficient permeation of withaferin-A, but not withanone, through normal cells. These data strongly validated our computational method for predicting permeability and hence bioavailability of candidate compounds in the drug development process.\n\nStatement of significanceWhat determines the bioavailability of a drug? Does the ability to cross cell membrane determine this? A combined simulation/experimental study of the permeability of two natural drugs - withanolides (Wi-A and Wi-N) across the cell membrane was conducted. In the computational portion of the study, steered MD simulations were performed to investigate the propensity of the two molecules to permeate across the cell. It is found that Wi-A proceeds relatively simply across the cell compared to Wi-N. This trend was found to be consistent with experiment. This work is an important step towards understanding the molecular basis of permeability of natural drug molecules.

biophysics↗

bioRxiv2018

Epigenetics of floral homeotic genes in relation to sexual dimorphism in the dioecious plant Mercurialis annua

In plants, dioecy characterizes species carrying male and female flowers on separate plants and occurs in about 6% of angiosperms. To date, the molecular mechanism(s) underlying sexual dimorphism is essentially unknown. The ability of gender-reversal by hormone application suggests that epigenetics might play an important role in sexual dimorphism. Proteome analysis of nuclei derived from flower buds of females, males and feminized males of the dioecious plant Mercurialis annua revealed differentially expressed proteins related to nucleic acid binding proteins, hydrolases and transcription factors, including floral homeotic genes. Further analysis showed that class B genes are mainly expressed in male flowers, while class D genes, as well as SUPERMAN-like genes, were mainly expressed in female flowers. Cytokinin-induced feminization of male plants was associated with down-regulation of male-specific genes concomitantly with up-regulation of female-specific genes. No correlation could be found between the expression of class B and D genes and their DNA methylation or chromatin conformation. Thus, our results ruled out epigenetic control over floral identity genes as the major determinants regulating sexual dimorphisms. Rather, determination of sex in M. annua might be controlled upstream of floral identity genes by a gender-specific factor that affects hormonal homeostasis.\n\nHighlightsSex determination in Mercurialis annua is not related to epigenetics of floral homeotic genes but appears to be modulated by an unknown gender-specific regulator(s) that affects hormonal homeostasis.

plant biology↗