Department of Biotechnology

Indian Institute of Technology Hyderabad (IITH) is a premier institute of science and technology established in 2008. IITH has been consistently ranked in the&nbsp;<a href="https://iith.ac.in/reports/" rel="noopener noreferrer">top 10 institutes</a>&nbsp;in India for Engineering&nbsp;<a href="https://www.nirfindia.org/2021/EngineeringRanking.html" rel="noopener noreferrer" target="_blank">according to NIRF</a>&nbsp;making it one of the most coveted schools for science and technology in the country.

IITH offers&nbsp;<a href="https://iith.ac.in/academics/under-graduate/">undergraduate programs</a>&nbsp;in all the classical engineering disciplines, applied sciences, design, as well as several modern interdisciplinary areas. Students are given a flexibility to explore a broad set of areas, and potentially pursue a minor or double major in a discipline that is not their own. Students who wish to seek a deeper understanding of their own discipline are strongly encouraged to get involved in cutting-edge research with the help of a faculty to mentor them, and earn an Honors in their own field.

The very foundation of IIT Hyderabad is based on&nbsp;<a href="https://iith.ac.in/research/">research and innovation</a>. The vibrant research culture is evident from the number of patents and publications that IITH produces consistently every year. IITH offers&nbsp;<a href="https://iith.ac.in/academics/programmes-offered/">graduate programs</a>&nbsp;at both a masters, and a doctoral level, in several diverse areas. There are separate programs for technology, design, science, and liberal arts. The&nbsp;<a href="https://iith.ac.in/academics/post-graduate/">MTech program</a>&nbsp;is offered in various modes and durations to cater to the ever-growing need of postgraduate level professionals in the country.

IITH encourages and supports&nbsp;<a href="https://iic-iith.netlify.app/index.html" rel="noopener noreferrer" target="_blank">innovation and entrepreneurship</a>&nbsp;at every stage. The minor program in entrepreneurship is open to all students of IITH, and many of the courses are offered by industrialists who are entrepreneurs themselves. There are various&nbsp;<a href="https://iith.ac.in/research/centres-incubators/">innovation centers and incubators</a>&nbsp;that promote entrepreneurship and help mentor young innovators. IITH has been very successful in building tie-ups with leading academic institutions around the globe.

IITH enjoys a very special relationship with Japanese universities and industries that goes beyond academic and research collaborations. In fact, some of the iconic buildings in IITH campus will carry the signature of Japanese architecture.

IITH creates a unique holistic ecosystem for education that offers interactive learning, a very flexible academic structure, cutting-edge research, strong industry collaboration, and entrepreneurship. This is an environment which enables students and faculty to translate their dreams into realities.

IIT Hyderabad

We present a technique to determine the orientation of single fluorophores attached to DNA origami structures based on two measurements. First, the orientation of the absorption transition dipole of the molecule is determined through a polarization-resolved excitation measurement. Second, the orientation of the DNA origami structure is obtained from a DNA-PAINT nanoscopy measurement. Both measurements are performed consecutively on a fluorescence wide-field microscope. We employed this approach to study the orientation of single ATTO 647N, ATTO 643, and Cy5 fluorophores covalently attached to a 2D rectangular DNA origami structure with different nanoenvironments, achieved by changing both the fluorophores' binding position and immediate vicinity. Our results show that when fluorophores are incorporated with additional space, for example, by omitting nucleotides in an elsewise double-stranded environment, they tend to stick to the DNA and to adopt a preferred orientation that depends more on the specific molecular environment than on the fluorophore type. With the aid of all-atom molecular dynamics simulations, we rationalized our observations and provide insight into the fluorophores' probable binding modes. We believe this work constitutes an important step toward manipulating the orientation of single fluorophores in DNA origami structures, which is vital for the development of more efficient and reproducible self-assembled nanophotonic devices. ©

World University root node

ACS Nano

Determining the In-Plane Orientation and Binding Mode of Single Fluorescent Dyes in DNA Origami Structures

The widespread use of triclosan in personal care products as an antimicrobial agent is leading to its alarming tissue-bioaccumulation including human brain. However, knowledge of its potential effects on the vertebrate nervous system is still limited. Here, we hypothesized that sublethal triclosan concentrations are potent enough to alter motor neuron structure and function in zebrafish embryos exposed for prolonged duration. In this study, zebrafish embryos were used as vertebrate-animal model. Prolonged exposure (up to 4 days) of 0.6 mg/L (LC50, 96 h) and 0.3 mg/L (&lt;LC50, Sublethal) triclosan produced aberrations in motor neuron innervations in skeletal muscles and reduced touch-evoked escape response in zebrafish larvae. This suggests motor dysfunction in treated embryos. To further explore the mechanisms of triclosan induced neurotoxicity, we determined the enzyme activity of acetylcholinesterase (AChE) and the expression of acetylcholinesterase (ache), myelin basic protein (mbp) and synapsin IIa (syn2a) genes which play an important role in the neural development and synaptic transmission. The ache and syn2a genes were down-regulated in triclosan treated larvae without any significant changes in mbp gene expression. At functional level, we observed a decrease in the AChE activity. Furthermore, docking results showed that triclosan can form a stable interaction with binding pocket of AChE and perhaps it can compete with natural acetylcholine for direct binding to AChE thereby inhibiting it and affecting cholinergic transmission. Therefore, triclosan can be regarded as a neurotoxic agent even at sublethal concentrations. Overall, the growing toxicological evidence against triclosan including ours suggest caution in its widespread use. © 2020 Elsevier Ltd

Chemosphere

Triclosan affects motor function in zebrafish larva by inhibiting ache and syn2a genes

Objective: Operative volume has been used as a marker of quality. Research from previous decades has suggested minimum open abdominal aortic aneurysm (AAA) repair volume requirements for surgeons of 9 to 13 open AAA repairs annually and for hospitals of 18 open AAA repairs annually to purportedly achieve acceptable results. Given concerns regarding the decreased frequency of open repairs in the endovascular era, we examined the association of surgeon and hospital volume with the 30- and 90-day mortality in the Vascular Quality Initiative (VQI) registry. Methods: Patients who had undergone elective open AAA repair from 2013 to 2018 were identified in the VQI registry. We performed a cross-sectional evaluation of the association between the average hospital and surgeon volume and 30-day postoperative mortality using a hierarchical Bayesian model. Cross-level interactions were permitted, and random surgeon- and hospital-level intercepts were used to account for clustering. The mortality results were adjusted by standardizing to the observed distribution of relevant covariates in the overall cohort. The outcomes were compared to the Society for Vascular Surgery guidelines recommended criteria of <5% perioperative mortality. Results: A total of 3078 patients had undergone elective open AAA repair by 520 surgeons at 128 hospitals. The 30- and 90-day risks of postoperative mortality were 4.1% (n = 126) and 5.4% (n = 166), respectively. The mean surgeon volume and hospital volume both correlated inversely with the 30-day mortality. Averaged across all patients and hospitals, we found a 96% probability that surgeons who performed an average of four or more repairs per year achieved <5% 30-day mortality. Substantial interplay was present between surgeon volume and hospital volume. For example, at lower volume hospitals performing an average of five repairs annually, <5% 30-day mortality would be expected 69% of the time for surgeons performing an average of three operations annually. In contrast, at higher volume hospitals performing an average of 40 repairs annually, a <5% 30-day mortality would be expected 96% of the time for surgeons performing an average of three operations annually. As hospital volume increased, a diminishing difference occurred in 30-day mortality between lower and higher volume surgeons. Likewise, as surgeon volume increased, a diminishing difference was found in 30-day mortality between the lower and higher volume hospitals. Conclusions: Surgeons and hospitals in the VQI registry achieved mortality outcomes of <5% (Society for Vascular Surgery guidelines), with an average surgeon volume that was substantially lower compared with previous reports. Furthermore, when considering the development of minimal surgeon volume guidelines, it is important to contextualize the outcomes within the hospital volumes. © 2020 Society for Vascular Surgery

Journal of Vascular Surgery

Association and interplay of surgeon and hospital volume with mortality after open abdominal aortic aneurysm repair in the modern era

Background: Myxobacteria harbor numerous biosynthetic gene clusters that can produce a diverse range of secondary metabolites. Minicystis rosea DSM 24000T is a soil-dwelling myxobacterium belonging to the suborderSorangiineae and family Polyangiaceae and is known to produce various secondary metabolites as well as polyunsaturated fatty acids (PUFAs). Here, we use whole-genome sequencing to explore the diversity of biosynthetic gene clusters in M. rosea. Results: Using PacBio sequencing technology, we assembled the 16.04 Mbp complete genome of M. rosea DSM 24000T, the largest bacterial genome sequenced to date. About 44% of its coding potential represents paralogous genes predominantly associated with signal transduction, transcriptional regulation, and protein folding. These genes are involved in various essential functions such as cellular organization, diverse niche adaptation, and bacterial cooperation, and enable social behavior like gliding motility, sporulation, and predation, typical of myxobacteria. A profusion of eukaryotic-like kinases (353) and an elevated ratio of phosphatases (8.2/1) in M. rosea as compared to other myxobacteria suggest gene duplication as one of the primary modes of genome expansion. About 7.7% of the genes are involved in the biosynthesis of a diverse array of secondary metabolites such as polyketides, terpenes, and bacteriocins. Phylogeny of the genes involved in PUFA biosynthesis (pfa) together with the conserved synteny of the complete pfa gene cluster suggests acquisition via horizontal gene transfer from Actinobacteria. Conclusion: Overall, this study describes the complete genome sequence of M. rosea, comparative genomic analysis to explore the putative reasons for its large genome size, and explores the secondary metabolite potential, including the biosynthesis of polyunsaturated fatty acids. © 2021, The Author(s).

Fulltext

BMC Genomics

Complete genome sequence and identification of polyunsaturated fatty acid biosynthesis genes of the myxobacterium Minicystis rosea DSM 24000T

As hypoxia is a major driver for the pathophysiology of COVID-19, it is crucial to characterize the hypoxic response at the cellular and molecular levels. In order to augment drug repurposing with the identification of appropriate molecular targets, investigations on therapeutics preventing hypoxic cell damage is required. In this work, we propose a hypoxia model based on alveolar lung epithelial cells line using chemical inducer, CoCl2 that can be used for testing calcium channel blockers (CCBs). Since recent studies suggested that CCBs may reduce the infectivity of SARS-Cov-2, we specifically select FDA approved calcium channel blocker, nifedipine for the study. First, we examined hypoxia-induced cell morphology and found a significant increase in cytosolic calcium levels, mitochondrial calcium overload as well as ROS production in hypoxic A549 cells. Secondly, we demonstrate the protective behaviour of nifedipine for cells that are already subjected to hypoxia through measurement of cell viability as well as 4D imaging of cellular morphology and nuclear condensation. Thirdly, we show that the protective effect of nifedipine is achieved through the reduction of cytosolic calcium, mitochondrial calcium, and ROS generation. Overall, we outline a framework for quantitative analysis of mitochondrial calcium and ROS using 3D imaging in laser scanning confocal microscopy and the open-source image analysis platform ImageJ. The proposed pipeline was used to visualize mitochondrial calcium and ROS level in individual cells that provide an understanding of molecular targets. Our findings suggest that the therapeutic value of nifedipine may potentially be evaluated in the context of COVID-19 therapeutic trials. © 2021

Department of Chemical Engineering

Free Radical Biology and Medicine

FDA approved L-type channel blocker Nifedipine reduces cell death in hypoxic A549 cells through modulation of mitochondrial calcium and superoxide generation

Human serum albumin (HSA) is an abundant carrier protein present in the blood plasma manifesting affinity for drugs and ligands. The bindings of several drugs can cause changes in the structural conformation of HSA that may affect its function. HSA is also known to in vitro form amyloid-like aggregates with fibrillar morphology as observed under TEM. Earlier, we showed that the HSA amyloid-like aggregates display self-seeding potential and detergent stability and the dimers of HSA, which are preferable for clinical applications due to their longer circulatory life, can also form amyloid-like aggregates. As aspirin, a commonly prescribed drug, was previously documented to acetylate HSA at one of its lysine residues, here, we examined if acetylation has any effect on the in vitro amyloid-like aggregation of HSA. We show that HSA acetylated in vitro using acetylsalicylic acid manifests relatively reduced levels of amyloid-specific properties such as turbidity, Thioflavin-T-positive aggregation, β-sheet content and stability against an ionic detergent. Also, TEM imaging shows that the acetylated HSA forms relatively less aggregates and with smaller sizes whereas, the aggregates of HSA are more abundant and larger in sizes with fibrillar morphology which further supports that acetylation can attenuate the amyloid-like aggregation of HSA. © 2021, National Institute of Science Communication and Information Resources. All rights reserved.

Indian Journal of Biochemistry and Biophysics

Pharmaceutical acetylation can modulate the amyloidogenicity of human serum albumin

BSA can form amyloid-like aggregates in vitro at 65 °C. Heterologous amyloid can proposedly cross-seed other protein's aggregation, however, general mechanisms and driving conditions remain to be vividly elucidated. Here, we examined if pre-formed HEWL amyloid can cross-seed the aggregation of BSA at physiological temperature, 37 °C, and whether the efficacy depends on the BSA conformation. We find that at pH 3.0, 37 °C where BSA manifests exposure of abundant hydrophobic patches, HEWL amyloid efficiently drives BSA into ThT-positive, sarkosyl-resistant, β-sheet rich amyloid-like aggregates exhibiting fibrils in TEM. On the contrary, HEWL amyloid fails to cross-seed the BSA aggregation at pH 7.0, 37 °C where BSA has largely internalized hydrophobic patches. Strikingly, human lysozyme amyloid could also cross-seed human serum albumin aggregation at pH 3.0, 37 °C. Thus, heterologous amyloid cross-seeding can help overcome the energy-barrier for aggregation of other proteins that, for any reason, may have perturbed and promiscuous structural conformation at physiological temperatures. © 2021 Elsevier B.V.

Biophysical Chemistry

Amyloid-like aggregation of bovine serum albumin at physiological temperature induced by cross-seeding effect of HEWL amyloid aggregates

The very chemical structure of DNA that enables biological heredity and evolution has non-trivial implications for the self-organization of DNA molecules into larger assemblies and provides limitless opportunities for building functional nanostructures. This progress report discusses the natural organization of DNA into chiral structures and recent advances in creating synthetic chiral systems using DNA as a building material. How nucleic acid chirality naturally comes into play in a diverse array of situations is considered first, at length scales ranging from an individual nucleotide to entire chromosomes. Thereafter, chiral liquid crystal phases formed by dense DNA mixtures are discussed, including the ongoing efforts to understand their origins. The report then summarizes recent efforts directed toward building chiral structures, and other structures of complex topology, using the principle of DNA self-assembly. Discussed last are existing and proposed functional man-made nanostructures designed to either probe or harness DNA's chirality, from plasmonics and spintronics to biosensing. © 2021 The Authors. Advanced Science published by Wiley-VCH GmbH

Advanced Science

Chiral Systems Made from DNA

Single-molecule imaging has gained momentum to quantify the dynamics of biomolecules in live cells, as it provides direct real-time measurements of various cellular activities under their physiological environment. Yeast, a simple and widely used eukaryote, serves as a good model system to quantify single-molecule dynamics of various cellular processes because of its low genomic and cellular complexities, as well as its facile ability to be genetically manipulated. In the past decade, significant developments have been made regarding the intracellular labeling of biomolecules (proteins, mRNA, fatty acids), the microscopy setups to visualize single-molecules and capture their fast dynamics, and the data analysis pipelines to interpret such dynamics. In this review, we summarize the current state of knowledge for the single-molecule imaging in live yeast cells to provide a ready reference for beginners. We provide a comprehensive table to demonstrate how various labs tailored the imaging regimes and data analysis pipelines to estimate various biophysical parameters for a variety of biological processes. Lastly, we present current challenges and future directions for developing better tools and resources for single-molecule imaging in live yeast cells. © 2021 Elsevier Ltd

Journal of Molecular Biology

In-vivo Single-Molecule Imaging in Yeast: Applications and Challenges

Non-CpG DNA methylation (non-mCpG) is enriched in the genome of brain neurons and germline cells in mammals. Accumulation of non-mCpG during postnatal brain development correlates with gene regulation and inactivation of distal regulatory elements. Recently, UHRF1 has been found to contribute to de novo non-CpG methylation, however, whether UHRF1 could recognize non-mCpG is unknown. Here, we have demonstrated through calorimetric measurements that the UHRF1 SRA can recognize mCpH and fully-mCpHpG, types of non-mCpG. Our ITC binding studies endorse the preferential reading of hemi-mCpG by UHRF1 SRA and also show 6-fold weaker binding for fully-mCpG than hemi-mCpG. Despite presence of symmetrical (5-methyl cytosine) 5mCs, stoichiometry of 1:1 for UHRF1 SRA binding to fully-mCpG indicates that UHRF1 SRA may not form a stable complex with fully-mCpG DNA. Contrarily, UHRF1 SRA recognizes fully-mCpHpG with a stoichiometry of 2:1 protein to DNA duplex with binding affinity higher than fully-mCpG. Our crystal structure of UHRF1 SRA bound to fully-mCpHpG DNA reveals dual flip-out mechanism of 5mC recognition. Metadynamics studies corroborates with ITC data that UHRF1 SRA could not form a stable complex with fully-mCpG DNA. Altogether, this study demonstrates that UHRF1 SRA recognizes non-mCpG DNA and exhibits contrasting mechanisms for hemi-mCpG and fully-mCpHpG DNA recognition. © 2018 Elsevier B.V.