Krishna Bhagavatula

Bio: Krishna Bhagavatula is an academic researcher from Agilent Technologies. The author has contributed to research in topics: Detection of genetically modified organisms & Globe. The author has an hindex of 2, co-authored 2 publications receiving 22 citations.

Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

Abstract: An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Abstract: Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry

Abstract: Owing to rapid growth in the elucidation of genome sequences of various organisms, deducing proteome sequences has become imperative, in order to have an improved understanding of biological processes. Since the traditional Edman method was unsuitable for high-throughput sequencing and also for N-terminus modified proteins, mass spectrometry (MS) based methods, mainly based on soft ionization modes: electrospray ionization and matrix-assisted laser desorption/ionization, began to gain significance. MS based methods were adaptable for high-throughput studies and applicable for sequencing N-terminus blocked proteins/peptides too. Consequently, over the last decade a new discipline called ‘proteomics’ has emerged, which encompasses the attributes necessary for high-throughput identification of proteins. ‘Proteomics’ may also be regarded as an offshoot of the classic field, ‘biochemistry’. Many protein sequencing and proteomic investigations were successfully accomplished through MS dependent sequence elucidation of ‘short proteolytic peptides (typically: 7–20 amino acid residues), which is called the ‘shotgun’ or ‘bottom-up (BU)’ approach. While the BU approach continues as a workhorse for proteomics/protein sequencing, attempts to sequence intact proteins without proteolysis, called the ‘top-down (TD)’ approach started, due to ambiguities in the BU approach, e.g., protein inference problem, identification of proteoforms and the discovery of posttranslational modifications (PTMs). The high-throughput TD approach (TD proteomics) is yet in its infancy. Nevertheless, TD characterization of purified intact proteins has been useful for detecting PTMs. With the hope to overcome the pitfalls of BU and TD strategies, another concept called the ‘middle-down (MD)’ approach was put forward. Similar to BU, the MD approach also involves proteolysis, but in a restricted manner, to produce ‘longer’ proteolytic peptides than the ones usually obtained in BU studies, thereby providing better sequence coverage. In this regard, special proteases (OmpT, Sap9, IdeS) have been used, which can cleave proteins to produce longer proteolytic peptides. By reviewing ample evidences currently existing in the literature that is predominantly on PTM characterization of histones and antibodies, herein we highlight salient features of the MD approach. Consequently, we are inclined to claim that the MD concept might have widespread applications in future for various research areas, such as clinical, biopharmaceuticals (including PTM analysis) and even for general/routine characterization of proteins including therapeutic proteins, but not just limited to analysis of histones or antibodies.

Antibody-free workflows for protein quantification by LC–MS/MS

Abstract: Antibody-free approaches for quantitative LC-MS/MS-based protein bioanalysis are reviewed and critically evaluated, and compared with the more widely used immunoaffinity-based approaches. Antibody-free workflows will be divided into four groups and discussed in the following order: direct analysis of signature peptides after proteolytic digestion; enrichment of target proteins and signature peptides by fractionated protein precipitation; enrichment of target proteins and signature peptides by reversed-phase and ion-exchange solid-phase extraction; and enrichment of target proteins and signature peptides by (antibody-free) affinity-solid-phase extraction.

Capillary Electrophoresis Based on Nucleic Acid Detection as Used in Food Analysis

Abstract: Food safety and food production are closely related to the health of consumers. Food-related accidents often cause tremendous losses of personnel and property. Thus, rapid detection and analysis of ingredients in food, tracing food sources, studying the optimal conditions for food production, and more are vital for preventing incidents related to safety. Conventional analysis based on proteomics, microbial cultures, and morphology, as well as biochemical tests based on metabonomics, are considered gold standards and used frequently, but they are labor-intensive, time-consuming, tedious, error-prone, and incapable of meeting the demand for rapid and precise detection at a large scale. Alternative detection methods that utilize capillary electrophoresis have the advantages of high efficiency, high throughput, high speed, and automation; these methods are coupled with various nucleic acid detection strategies to overcome the drawbacks of traditional identification methods, and to prevent false results. Therefore, this review focuses on the application of capillary electrophoresis based on nucleic acid detection in food analysis and provides an introduction to the limitations, advantages, and future developments of this approach.