Biology subjects

Bari, S.

Publications and source records attributed to Bari, S..

3 recordsLinked to original sources

bioRxiv2025

X-ray Spectroscopy Meets Native Mass Spectrometry: Probing Gas-phase Protein Complexes

Gas-phase activation and dissociation studies of biomolecules, proteins and their non-covalent complexes using X-rays hold great promise for revealing new insights into the structure and function of biological samples. This is due to the unique properties of X-ray molecular interactions, such as site-specific and rapid ionization. In this perspective, we report and discuss the promise of first proof-of-principle studies of X-ray-induced dissociation of native biological samples ranging from small 17 kDa monomeric proteins up to large 808 kDa non-covalent protein assemblies conducted at a synchrotron (PETRA III) and a free-electron laser (FLASH2). A commercially available quadrupole time-of-flight mass spectrometer (Q-ToF2, Micromass/Waters), modified for high-mass analysis by MS Vision, was further adapted for integration with the open ports at the corresponding beamlines. The protein complexes were transferred natively into the gas phase via nano-electrospray ionization and subsequently probed by extreme ultraviolet (FLASH2) or soft X-ray (PETRA III) radiation, in either their folded state or following collision-induced activation in the gas phase. Depending on the size of the biomolecule and the activation method, protein fragmentation, dissociation, or enhanced ionization were observed. Additionally, an extension of the setup by ion mobility is described, which can serve as a powerful tool for structural separation of biomolecules prior to X-ray probing. The first experimental results are discussed in the broader context of current and upcoming X-ray sources, highlighting their potential for advancing structural biology in the future.

biochemistry↗

bioRxiv2023

Nanobodies against SARS-CoV-2 reduced virus load in the brain of challenged mice and neutralized Wuhan, Delta and Omicron Variants

In this work, we developed llama-derived nanobodies (Nbs) directed to the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Nanobodies were selected after the biopanning of two VHH-libraries, one of which was generated after the immunization of a llama (lama glama) with the bovine coronavirus (BCoV) Mebus, and another with the full-length pre-fused locked S protein (S-2P) and the RBD from the SARS-CoV-2 Wuhan strain (WT). Most of the neutralizing Nbs selected with either RBD or S-2P from SARS-CoV-2 were directed to RBD and were able to block S- 2P/ACE2 interaction. Three Nbs recognized the N-terminal domain (NTD) of the S-2P protein as measured by competition with biliverdin, while some non-neutralizing Nbs recognize epitopes in the S2 domain. One Nb from the BCoV immune library was directed to RBD but was non-neutralizing. Intranasal administration of Nbs induced protection ranging from 40% to 80% against COVID-19 death in k18-hACE2 mice challenged with the WT strain. Interestingly, protection was not only associated with a significant reduction of virus replication in nasal turbinates and lungs, but also with a reduction of virus load in the brain. Employing pseudovirus neutralization assays, we were able to identify Nbs with neutralizing capacity against the Alpha, Beta, Delta and Omicron variants. Furthermore, cocktails of different Nbs performed better than individual Nbs to neutralize two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest these Nbs can potentially be used as a cocktail for intranasal treatment to prevent or treat COVID-19 encephalitis, or modified for prophylactic administration to fight this disease.

immunology↗

bioRxiv2020

Ethanol tolerance in Escherichia coli DH5-Alpha developed by serial exposure to sublethal doses is conferred to wild strains by horizontal gene transfer

Microorganisms evolve novel mechanisms and pathways to mitigate various stresses. These are developed by the accumulation of beneficial mutations over many generations. Such adaptations are often transferred from mutant microorganisms that develop it intrinsically to wild ones through horizontal gene transfer (HGT). It allows the latter to acquire favourable traits without having to employ resources to natively evolve. We ascertained this in Escherichia coli by first developing tolerance to ethanol, a potent disinfectant, by laboratory evolution and then transmitting it to the wild strain by HGT. Naturally, wild type E. coli cannot survive beyond 35% v/v ethanol in LB media. By serially increasing the concentration of ethanol by 5% v/v and selecting the surviving colonies, we were able to impose an artificial selection pressure. This in vitro microevolution increased the ethanol tolerance in our mutant to 75% v/v. To test if this tolerance could be transferred to the wild strain through HGT, we meticulously exposed the ancestral wild E. coli to the newly tolerant mutants to facilitate the exchange of genetic material. After our exposure, the unadapted wild type E. coli acquired tolerance up to 55% v/v through transformation and 45% v/v through transduction. The baseline tolerance of 35% v/v remained unchanged after conjugation. While our results are still preliminary, they provide interesting insights into the role horizontal gene transfer in developing resistance to bacteriocidal stressors.

microbiology↗