Biology subjects

SHARMA, P.

Publications and source records attributed to SHARMA, P..

3 recordsLinked to original sources

bioRxiv2024

Method for isolation of small extracellular vesicles from different biofluids and workflow for Mass Spectrometry based-shotgun proteomics and RNA isolation

Small extracellular vesicles (sEVs) or exosomes are small-sized (30-150 nm), nanoparticles that are released from almost all cells under normal and pathophysiological conditions. The sEVs have a vital role in biological systems as they communicate and transfer their contents, such as proteins, lipids, and nucleic acids, from the cells of origin to nearby or distant cells. In recent years, there has been a growing interest in isolating sEVs for use in disease mechanisms, clinical diagnoses, and therapeutics. Due to their small size sEVs can be observed using electron microscopy. The size distribution and concentration were checked by Nanoparticle Tracking Analysis. Western blotting confirmed the presence of exosome markers. The ease of obtaining patient samples from biofluids like plasma, saliva, and urine makes them a valuable source for diagnostic purposes by isolating sEVs to diagnose and predict diseases early. However, there is no specific protocol to perform it altogether. We have developed an improved ultracentrifugation method using gradient ultracentrifugation and ultrafiltration, which results in higher sEVs purity and yield. We have tested this method on plasma, saliva, and urine at a single platform, and we have isolated proteins and RNA from exosomes for their downstream applications. Our method is simple to use and can be utilized for clinical research biomarker applications, in understanding disease mechanisms and monitoring its progressions from biofluid sample collections.

cell biology↗

bioRxiv2024

Methodology for isolation of serum, cerebrospinal fluid, and hippocampal neuron proteins from rat and their analysis using mass spectrometry-based shotgun proteomics

Emerging interests in the field of research related to diseases such as Alzheimers disease, Parkinsons disease, cognition, and other mental health-related disorders have prompted a need for a common method for the isolation of serum, CSF, and hippocampus. The hippocampus is responsible for learning and memory. It can be affected by various neurological and psychiatric disorders. However, the process of collecting samples such as CSF and hippocampal neurons is challenging, especially for small animals like rats. We have presented here a method for the isolation of serum, CSF, and hippocampal neurons that can be used for its downstream applications such as proteomics. We have used high-speed centrifugation instruments and density gradient centrifugation methods, which are easy to follow. Additionally, we have tested the proteins identified through mass spectrometry. Our method enables the study of proteins in serum, CSF, and neural cells for researching protein cross-talks and neurological disorder mechanisms.

neuroscience↗

bioRxiv2023

Stumpy forms are the predominant transmissible forms of Trypanosoma brucei.

Schuster et al. demonstrated that bloodstream slender forms of African trypanosomes are readily transmissible to young tsetse flies where they can complete their complex life cycle (1). In their experimental conditions, a single slender parasite was sufficient for productive infection. Here, we compared the infectivity of slender and stumpy bloodstream forms in adult flies with a mature immune system, and without using any chemical compounds that would alter the insect immune response and/or promote the infection. After ingestion of slender forms, infected flies were observed only in one out of 24 batches of non-immunocompetent teneral flies and with a high number of parasites. In contrast, infected flies were detected in 75% (18/24) of the batches infected with stumpy parasites, and as few as 10 stumpy parasites produced mature infections in immune adult flies. We discuss that, although Schuster et al. have demonstrated the intrinsic capacity of slender form trypanosomes to infect young and naive tsetse flies, highlighting the remarkable plasticity and adaptability of these protists, this phenomenon is unlikely to significantly contribute to the epidemiology of African trypanosomiases. According to both experimental and field observations, stumpy forms appear to be the most adapted forms for African trypanosome transmission from the mammalian host to the tsetse fly vector in natural conditions.

microbiology↗