Chromatin modifications affect several processes. In investigating the Leishmania donovani histone acetyltransferase HAT2, using in vitro biochemical assays and HAT2-heterozygous genomic knockout we found the constitutively nuclear HAT2 acetylated histone H4K10 in vitro and in vivo. HAT2 was essential. HAT2-depleted cells displayed growth and cell cycle defects, and poor survival in host cells. Real time PCR and DNA microarray analyses, as well as rescue experiments, revealed that downregulation of cyclins CYC4 and CYC9 were responsible for S phase and G2/M defects of HAT2-depleted cells respectively. Leishmania genes are arranged in unidirectional clusters, and clustered genes are coordinately transcribed as long polycistronic units, typically from divergent strand switch regions (dSSRs) which initiate transcription bidirectionally on opposite strands. In investigating the mechanism by which CYC4 and CYC9 expression levels are reduced in HAT2-depleted cells without other genes in their polycistronic transcription units being coordinately downregulated, we found using reporter assays that CYC4 and CYC9 have their own specific promoters. Chromatin immunoprecipitation assays with H4acetylK10 antibodies and real time PCR analyses of RNA suggested these gene-specific promoters were activated in cell cycle-dependent manner. Nuclear run-on analyses confirmed that CYC4 and CYC9 were transcriptionally activated from their own promoters at specific cell cycle stages. Thus, there are two tiers of gene regulation. Transcription of polycistronic units primarily initiates at dSSRs, and this most likely occurs constitutively. A subset of genes have their own promoters, at least some of which are activated in a cell-cycle dependent manner. This second tier of regulation is more sensitive to H4K10 acetylation levels, resulting in downregulation of expression in HAT2-depleted cells. This report presents the first data pointing to cell cycle-specific activation of promoters in trypanosomatids, thus uncovering new facets of gene regulation in this parasite family.

Histone acetyltransferases impact multiple processes. This study investigates the role of histone acetyltransferase HAT4 in Leishmania donovani. Though HAT4 was dispensable for survival, its elimination decreased cell viability and caused cell cycle defects, with HAT4-nulls experiencing an unusually long G2/M. Survival of HAT4-nulls in macrophages was also substantially compromised. DNA microarray analysis revealed that HAT4 modestly regulated the expression of only a select number of genes, thus not being a major modulator of global gene expression. Significantly, cdc20 was among the downregulated genes. To ascertain if decreased expression of cdc20 was responsible for HAT4-null growth and cell cycle defects we expressed LdCdc20 ectopically in HAT4-nulls. We found this to alleviate the aberrant growth and cell cycle progression patterns displayed by HAT4-nulls, with cells navigating G2/M phase and re-entering G1 phase smoothly. HAT4-nulls expressing LdCdc20 ectopically showed survival rates comparable to wild type within macrophages, suggesting that G2/M defects were responsible for poor survival of HAT4-nulls within host cells also. These are the first data analyzing the in vivo functional role of HAT4 in any trypanosomatid. Our results directly demonstrate for the first time a role for Cdc20 in regulating trypanosomatid G2/M events, opening avenues for further research in this area.

Background: COVID-19 pandemic has caused an extraordinary situation, especially for the healthcare workers (HCWs), leading to increased psychological stress. The aim of the study was to estimate the prevalence of different grades of anxiety and depression across different centers in the Philippines and identify demographic factors associated with them. Design and Method: A cross-sectional, web-based, multi-center study was conducted among HCWs of Philippines from April 20- May 20, 2020. The study instruments used were the Generalized Anxiety Disorder (GAD-7) scale and Patient Health Questionnaire (PHQ-9). Risk perception scores were analyzed using Mann-Whitney and Kruskal-Wallis test. Logistic regression was done to identify factors significantly associated with symptoms of anxiety and depression determined. Results: A total of 516 HCWs were included in the study. Most of them have anxiety symptoms (70.74%), but only half of them have symptoms of depression (50.97%). In addition, gender, age, marital status, living status, occupation, work premises, and availability of mental health services were significantly associated with the participants’ anxiety symptoms; In contrast, gender, marital status, occupation, and work premises were significantly associated with depression symptoms. Conclusion: This study reiterates the fact and demonstrates that COVID-19 has disrupted the mental well-being of HCWs in the Philippines. Majority of HCW was psychologically affected by COVID-19. Therefore, there is a dire need to address mental illness amongst HCWs and frame guidelines based on proven algorithms to overcome these mental illnesses.

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.

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 ...