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The members of DHH superfamily have been reported with diverse substrate spectrum and play pivotal roles in replication, repair, and RNA metabolism. This family comprises phosphatases, phosphoesterase and bifunctional enzymes having nanoRNase and phosphatase activities. Cell cycle factor Cdc45, a member of this superfamily, is crucial for movement of the replication fork during DNA replication and an important component of the replisome. The specific protein-protein interactions of Cdc45 with other factors along with helicase moderate the faithful DNA replication process. However, the exact biochemical functions of this factor are still unknown and need further investigation. Here, we studied the biochemical roles of Cdc45 and its molecular interactions within the replisomal complex. The alteration in the level of protein, observed when DNA damage is induced in-vivo, suggests its association with DNA replication stress. We analyzed protein Cdc45, providing new insights about the molecular and biochemical functionality of this replisomal factor.

The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic monoclonal antibodies (mAbs). Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concern (VOCs) with high efficiency, including the highly transmissible Omicron. The crystal structure of the P4A2 Fab:RBD complex revealed that the residues of the RBD that interact with P4A2 are a part of the ACE2-receptor-binding motif and are not mutated in any of the VOCs. The pan coronavirus pseudotyped neutralization assay confirmed that the P4A2 mAb is specific for SARS-CoV-2 and its VOCs. Passive administration of P4A2 to K18-hACE2 transgenic mice conferred protection, both prophylactically and therapeutically, against challenge with VOCs. Overall, our data shows that, the P4A2 mAb has immense therapeutic potential to neutralize the current circulating VOCs. Due to the overlap between the P4A2 epitope and ACE2 binding site on spike-RBD, P4A2 may also be highly effective against a number of future variants.

Phage display is a proven and widely used technology for selecting specific antibodies against desired targets. However, an immense amount of effort is required to identify and screen the desired positive clones from large and diverse combinatorial libraries. On the other hand, the selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias toward clones with randomly produced amber stop codons, making it more difficult to identify desirable binding antibodies. To overcome the screening of desired clones with amber codons, we present a step-by-step approach for effective phage library screening to isolate useful antibodies. The procedure calls for creating a simple new vector system for soluble production of phage ELISA positive binding clones with one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences, which is otherwise difficult in standard screening.Graphical abstract:

ABSTRACT The worldwide increase in the frequency of multidrug-resistant and extensively drug-resistant cases of tuberculosis is mainly due to therapeutic noncompliance associated with a lengthy treatment regimen. Depending on the drug susceptibility profile, the treatment duration can extend from 6 months to 2 years. This protracted regimen is attributed to a supposedly nonreplicating and metabolically inert subset of the Mycobacterium tuberculosis population, called “persisters.” The mechanism underlying stochastic generation and enrichment of persisters is not fully known. We have previously reported that the utilization of host cholesterol is essential for mycobacterial persistence. In this study, we have demonstrated that cholesterol-induced activation of a RNase toxin (VapC12) inhibits translation by targeting proT tRNA in M. tuberculosis. This results in cholesterol-specific growth modulation that increases the frequency of generation of the persisters in a heterogeneous M. tuberculosis population. Also, a null mutant strain of this toxin (ΔvapC12) demonstrated an enhanced growth phenotype in a guinea pig model of M. tuberculosis infection, depicting its role in disease persistence. Thus, we have identified a novel strategy through which cholesterol-specific activation of a toxin-antitoxin module in M. tuberculosis enhances persister formation during infection. The current findings provide an opportunity to target persisters, a new paradigm facilitating tuberculosis drug development. IMPORTANCE The current TB treatment regimen involves a combination of drugs administered for an extended duration that could last for 6 months to 2 years. This could lead to noncompliance and the emergence of newer drug resistance strains. It is widely perceived that the major culprits are the so-called nonreplicating and metabolically inactive “persister” bacteria. The importance of cholesterol utilization during the persistence stage of M. tuberculosis infection and its potential role in the generation of persisters is very intriguing. We explored the mechanism involved in the cholesterol-mediated generation of persisters in mycobacteria. In this study, we have identified a toxin-antitoxin (TA) system essential for the generation of persisters during M. tuberculosis infection. This study verified that M. tuberculosis strain devoid of the VapBC12 TA system failed to persist and showed a hypervirulent phenotype in a guinea pig infection model. Our studies indicate that the M. tuberculosis VapBC12 TA system acts as a molecular switch regulating persister generation during infection. VapBC12 TA system as a drug target offers opportunities to develop shorter and more effective treatment regimens against tuberculosis.

Hepatitis E virus (HEV) is associated with acute hepatitis disease, which may lead to chronic disease in immunocompromised individuals. The disease is particularly severe among pregnant women (20–30% mortality). The only licensed vaccine against HEV, which is available in China, is the Escherichia coli purified recombinant virus-like particles (VLPs) encompassing the 368–660 amino acids (aa) of the viral ORF2 protein. The viral capsid is formed by the ORF2 protein, which harbors three glycosylation sites. Baculo virus expression system has been employed to generate a glycosylated VLP, which encompasses 112–608aa of the ORF2 protein. Here, we sought to produce a recombinant VLP containing 112–608aa of the ORF2 protein in Pichia pastoris (P. pastoris) expression system. The cDNA sequence encoding 112–608aa of the ORF2 protein was fused with the α-mating factor secretion signal coding sequence (for release of the fusion protein to the culture medium) and cloned into the yeast vector pPICZα. Optimum expression of recombinant protein was obtained at 72 h induction in 1.5% methanol using inoculum density (A600) of 80 and at pH-3.0 of the culture medium. Identity of the purified protein was confirmed by mass spectrometry analysis. Further studies revealed the glycosylation pattern and VLP nature of the purified protein. Immunization of BALB/c mice with these VLPs induced potent immune response as evidenced by the high ORF2 specific IgG titer and augmented splenocyte proliferation in a dose dependent manner. 112–608aa ORF2 VLPs produced in P. pastoris appears to be a suitable candidate for development of diagnostic and prophylactic reagents against the hepatitis E.