Srishti Joshi

Herein, a step-by-step protocol for simultaneous detection of 20 amino acids commonly present in cell culture media is described. The protocol facilitates detection of both primary and secondary am...

We report a structure-based approach to design peptides that can bind to aggregation prone, partially folded intermediates (PFI) of insulin, thereby inhibiting early stages of aggregation nucleation. We account for the important role of modular architecture of protein-protein binding interfaces and tertiary structure heterogeneity of the PFIs in design of peptide inhibitors. Determination of association hot spots revealed that two interface segments are required to capture majority contribution to insulin homodimer binding energy. Selection of peptides that will have a high probability to inhibit insulin self-association was done on the basis of similarity in binding interface coverage of PFI residues in the peptide-PFI complex and the native-PFI dimer. Data on aggregate growth rate and secondary structure for formulations incubated under amyloidogenic conditions show that designed peptides inhibit insulin aggregation in a concentration dependent manner. Mechanism of aggregation inhibition was probed by determining enthalpy of peptide-insulin binding and peptide micellization using isothermal titration calorimetry. Finally, effect of designed peptides on insulin activity was quantified using a spectrophotometric assay for glucose uptake by HepG2 cells.

Biotherapeutics are protein products generated using recombinant DNA technology and manufactured in prokaryotic or eukaryotic cells. It is often said that “the process is the product” and thereby the effect of the manufacturing process is etched on the final product in the form of its heterogeneity. For any biotherapeutic, the acceptable range of the critical quality attributes is defined based on the expected impact of a specific variation on the product stability, safety, and efficacy. For a biosimilar to receive regulatory approval, the manufacturer must demonstrate analytical and clinical comparability with the originator product. As this is mandatory, every biosimilar manufacturer performs this exercise for each biosimilar product under development. However, few reports of thorough evaluation of the quality of biosimilar products are available in the literature.

Bulb onion ( Allium cepa L.) is a challenging subject for experimental studies because of its slow growth, genetic heterogeneity, and sensitivity to environmental and biotic stresses. Sharing of common germplasm and controlled propagation practices has underpinned research on model plants, such as Arabidopsis and tomato, but not in onion. To encourage wider evaluation of onion for physiological and molecular studies in controlled environments, we describe the growing practices we have developed over two decades of research on adaptive and nutrient assimilation traits. Key aspects covered include choice of germplasm, propagation media, nutrition, and environmental control. Adopting common onion genetics and cultivation techniques across laboratories will allow researchers to answer deeper research questions and increase the reproducibility of the research.