Just when the world started to adapt to the ‘new normal’ amid the coronavirus disease 19 (COVID‐19) pandemic, the world is witnessing the wrath of another viral disease, the monkeypox virus (MPXV). The virus is endemic to African countries, where several outbreaks have been reported in the past. However, the present cases have been reported in non‐endemic countries worldwide. Although MPX is considered to be a self‐limiting disease, recent reports on its incidence have proved otherwise. The 2022 multi‐country MPX outbreak has drawn the attention of global surveillance organizations and epidemiologists to trace its origin; however, there are existing gaps regarding the animal reservoirs, biological implications, and management of MPX. In view of the recent events, the World Health Organization (WHO) has also declared the ongoing MPX outbreak a global health emergency. Hence, the geographically expanding MPXV poses a significant threat to human health and public safety. In this review, the latest insights into the biology of MPXV have been provided by discussing its biological implications on human health, changing epidemiological footprint, and presently available intervention strategies. This review also sheds light on the existing lacunas and possible reasons that may have been responsible for the ongoing MPX outbreak.

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, also interacting with each other directly in vitro. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival here as well. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins that associate with PCNA have been identified in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it may not be confined to trypanosomatids.

DNA methylation events mediated by orphan methyltransferases modulate various cellular processes like replication, repair and transcription. Bacteria and archaea also harbor DNA methyltransferases that are part of restriction-modification systems, which serve to protect the host genome from being cleaved by the cognate restriction enzyme. While DNA methylation has been exhaustively investigated in bacteria it remains poorly understood in archaea. Picrophilus torridus is a euryarchaeon that can thrive under conditions of extremely low pH (0.7), and thus far no reports have been published regarding DNA methylation in this extremophile. This study reports the first experimentation examining DNA methylation in P. torridus. We find the genome to carry methylated adenine (m6A) but not methylated cytosine (m5C) residues. The m6A modification is absent at GATC sites, indicating the absence of an active Dam methylase even though the dam gene has been annotated in the genome sequence. Two other methylases have also been annotated in the P. torridus genome sequence. One of these is a part of a Type I restriction-modification system. Considering that all Type I modification methylases characterized to date target adenine residues, the modification methylase of this Type I system has been examined. The genes encoding the S subunit (that is responsible for DNA recognition) and M subunit (that is responsible for DNA methylation) have been cloned and the recombinant protein purified from E.coli, and regions involved in M-S interactions have been identified. The M.PtoI enzyme harbors all the motifs that typify Type I modification methylases, and displays robust adenine methylation in in vitro assays under a variety of conditions. Interestingly, magnesium is essential for enzyme activity. The enzyme displays substrate inhibition at higher concentrations of AdoMet. Mutational analyses reveal that Motif I plays a role in AdoMet binding, and Motif IV is critical for methylation activity. The data presented here lays the foundation for further research in the area of DNA methylation and restriction-modification research in this most unusual microorganism.

29DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase 30 whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in 31Leishmania donovani Cdc45. This motif is typically linked to interaction with the 32 eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can 33 potentially bind upto three different proteins simultaneously via a loop region present in 34 each monomer. Multiple binding partners have been identified from among the replication 35 machinery in other eukaryotes, and the concerted /sequential binding of these partners are 36 central to the fidelity of the replication process. Though conserved in Cdc45 across 37Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei 38Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such 39 an interaction in the in vivo context. Having confirmed the importance of Cdc45 in 40Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole 41 cell extracts, interacting with each other directly in vitro also. The interaction is mediated 42 via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance 43 of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to 44 be essential for cell survival in this organism also. Our results implicate a role for the 45 Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-46 PCNA interaction might help tether PCNA and associated replicative DNA polymerase to 47 Author Summary: 54Leishmaniases are manifested in three forms: cutaneous, sub-cutaneous and visceral. The 55 prevalent form in the Indian subcontinent is visceral Leishmaniasis (VL), which is fatal if 56 not treated on time. While there are drugs for treatment, the hunt for additional drugs 57 continues due to emerging drug resistance patterns. The parasite is transmitted by the bite 58 of the sandfly, whereupon it establishes itself within cells of the host immune system 59 (macrophages) and reproduces by binary fission. The replication of its genome is essential 60 for parasite survival. Eukaryotic DNA replication is generally conserved across species. 61This study targets Cdc45, a protein that helps unwind the DNA double helix to enable 62 copying of the two strands into two daughter strands. The new chains of DNA are 63 synthesized by DNA polymerases, and a trimeric protein, proliferating cell nuclear antigen 64 (PCNA), helps clamp the polymerases onto the template. In this study we find Cdc45 to 65 interact with PCNA, and have identified the motif in Cdc45 via which it does so. Our 66 results suggest this interaction is seen in some other eukaryotes as well. Based on the 67 results of our experiments we propose that Cdc45 may help moor PCNA-polymerase 68 complexes to template DNA. 69 70 71 72 73 74 75 76 77 78 HIV-Leishmania infections, and is deadly if not treated in a timely manner. In the Indian 83 subcontinent ...

The monkeypox virus (MPXV) has become a major threat due to the increasing global caseload and the ongoing multi-country outbreak in non-endemic territories. Due to limited research in this avenue and the lack of intervention strategies, the present study was aimed to virtually screen bioactive phytochemicals against envelope proteins of MPXV via rigorous computational approaches. Molecular docking and molecular dynamic (MD) simulations were used to investigate the binding affinity of 12 phytochemicals against three envelope proteins of MPXV, viz., D13, A26, and H3. Silibinin, oleanolic acid, and ursolic acid were computationally identified as potential phytochemicals that showed strong binding affinity towards all the tested structural proteins of MPXV through molecular docking. The stability of the docked complexes was also confirmed by MD simulations. ADME analysis also computationally confirmed the drug-like properties of the phytochemicals, thereby asserting their suitability for consumption. Hence, this study envisions the candidature of bioactive phytochemicals as promising inhibitors against the MPXV, serving as template molecules that could further be experimentally evaluated for their efficacy against monkeypox.