Trypanosoma brucei BRCA2 in the regulation of genome stability and DNA repair

Trypanosoma brucei is a protistan parasite of mammals that evades its host’s immune responses by antigenic variation, which in T. brucei involves the periodic switching of the Variant Surface Glycoprotein (VSG) coat to antigenically distinct variants. The T. brucei genome contains a huge archive of silent VSG genes that are expressed from specialised expression sites, only one of which is actively transcribed at any one time. Copying of silent VSG genes into the active expression site has been shown to occur by homologous recombination, as mutation of the RAD51 recombinase and a distantly related gene, RAD51-3, impairs this process. BRCA2 is a protein that binds and regulates the function of Rad51 during homologous recombination. Mutation of BRCA2 in bloodstream form T. brucei leads to increased sensitivity to DNA damaging agents, and impairments in homologous recombination, RAD51 subnuclear foci formation and VSG switching, suggesting that it too acts in recombination-repair and antigenic variation. Beyond these phenotypes, an accumulation of putative gross chromosomal rearrangements in the megabase chromosomes of the T. brucei genome and a novel replication phenotype were also observed, and the basis of both these processes was unclear. T. brucei BRCA2 is highly unusual relative to orthologues in other eukaryotes, as the protein contains an expansion in the number of RAD51-binding BRC repeat motifs, which are arranged in a tandem repeat array that has not been observed elsewhere. In order to examine the function of BRCA2 in the maintenance of genome stability in T. brucei, brca2 homozygous mutants were generated in procyclic form TREU 927 and Lister 427 cells. Analysis of genomic stability by Southern blotting and pulsed field agarose gel electrophoresis revealed that BRCA2’s function in the maintenance of genome stability appears to be either bloodstream form-specific, or plays a more substantial role in this life cycle stage. To examine the function of the BRC repeat expansion, cell lines containing variants of BRCA2 with reduced numbers of BRC repeats were generated, expressed in brca2 homozygous mutants and phenotype analysis carried out. Growth and DNA repair were restored by the expression of virtually all variants, suggesting the BRC repeat expansion is not an adaptation for general genome maintenance, though the repair activity of a variant with a single BRC repeat appeared to differ between bloodstream and procyclic form parasites. In contrast to this, a striking correlation between BRC repeat number and the regulation of RAD51 subnuclear dynamics was observed, showing that the BRC array expansion has important functional significance. GST pull-down analysis was used to examine the domains of T. brucei BRCA2 that interact with RAD51, revealing an extent of interaction not apparent in BRCA2 orthologues in other organisms. This complexity of interaction was further analysed by immunolocalisation of BRCA2 and RAD51, before and after DNA damage, which showed potentially dynamic co-localisation of the two repair factors. Finally, a putative interaction between T. brucei BRCA2 and CDC45 was tested both in vitro and in vivo, but could not be validated, suggesting it does not provide an explanation for the replication defects observed in bloodstream form brca2-/- mutant cells. All of the analyses above shed light on the function of the BRCA2 protein in the regulation of homologous recombination in T. brucei

Genome-wide screens connect HD82 loss-of-function to purine analog resistance in African trypanosomes

Nucleoside analogs have been used extensively as anti-infective agents, particularly against viral infections, and have long been considered promising anti-parasitic agents. These pro-drugs are metabolized by host-cell, viral, or parasite enzymes prior to incorporation into DNA, thereby inhibiting DNA replication. Here, we report genes that sensitize African trypanosomes to nucleoside analogs, including the guanosine analog, ganciclovir. We applied ganciclovir selective pressure to a trypanosome genome-wide knockdown library, which yielded nucleoside mono- and diphosphate kinases as hits, validating the approach. The two most dominant hits to emerge, however, were Tb927.6.2800 and Tb927.6.2900, which both encode nuclear proteins; the latter of which is HD82, a SAMHD1-related protein and a putative dNTP triphosphohydrolase. We independently confirmed that HD82, which is conserved among the trypanosomatids, can sensitize Trypanosoma brucei to ganciclovir. Since ganciclovir activity depends upon phosphorylation by ectopically expressed viral thymidine kinase, we also tested the adenosine analog, ara-A, that may be fully phosphorylated by native T. brucei kinase(s). Both Tb927.6.2800 and HD82 knockdowns were resistant to this analog. Tb927.6.2800 knockdown increased sensitivity to hydroxyurea, while dNTP analysis indicated that HD82 is indeed a triphosphohydrolase with dATP as the preferred substrate. Our results provide insights into nucleoside/nucleotide metabolism and nucleoside analog metabolism and resistance in trypanosomatids. We suggest that the product of 6.2800 sensitizes cells to purine analogs through DNA repair, while HD82 does so by reducing the native purine pool.IMPORTANCEThere is substantial interest in developing nucleoside analogs as anti-parasitic agents. We used genome-scale genetic screening and discovered two proteins linked to purine analog resistance in African trypanosomes. Our screens also identified two nucleoside kinases required for pro-drug activation, further validating the approach. The top novel hit, HD82, is related to SAMHD1, a mammalian nuclear viral restriction factor. We validated HD82 and localized the protein to the trypanosome nucleus. HD82 appears to sensitize trypanosomes to nucleoside analogs by reducing native pools of nucleotides, providing insights into both nucleoside/nucleotide metabolism and nucleoside analog resistance in trypanosomatids

Post-transcriptional reprogramming by thousands of mRNA untranslated regions in trypanosomes

Although genome-wide polycistronic transcription places major emphasis on post-transcriptional controls in trypanosomatids, messenger RNA cis-regulatory untranslated regions (UTRs) have remained largely uncharacterised. Here, we describe a genome-scale massive parallel reporter assay coupled with 3’-UTR-seq profiling in the African trypanosome and identify thousands of regulatory UTRs. Increased translation efficiency was associated with dosage of adenine-rich poly-purine tracts (pPuTs). An independent assessment of native UTRs using machine learning based predictions confirmed the robust correspondence between pPuTs and positive control, as did an assessment of synthetic UTRs. Those 3’-UTRs associated with upregulated expression in bloodstream-stage cells were also enriched in uracil-rich poly-pyrimidine tracts, suggesting a mechanism for developmental activation through pPuT ‘unmasking’. Thus, we describe a cis-regulatory UTR sequence ‘code’ that underpins gene expression control in the context of a constitutively transcribed genome. We conclude that thousands of UTRs post-transcriptionally reprogram gene expression profiles in trypanosomes

Genome-wide screens connect HD82 loss-of-function to purine analog resistance in African trypanosomes

Nucleoside analogs have been used extensively as anti-infective agents, particularly against viral infections, and have long been considered promising anti-parasitic agents. These pro-drugs are metabolized by host-cell, viral, or parasite enzymes prior to incorporation into DNA, thereby inhibiting DNA replication. Here, we report genes that sensitize African trypanosomes to nucleoside analogs, including the guanosine analog, ganciclovir. We applied ganciclovir selective pressure to a trypanosome genome-wide knockdown library, which yielded nucleoside mono- and diphosphate kinases as hits, validating the approach. The two most dominant hits to emerge, however, were Tb927.6.2800 and Tb927.6.2900, which both encode nuclear proteins; the latter of which is HD82, a SAMHD1-related protein and a putative dNTP triphosphohydrolase. We independently confirmed that HD82, which is conserved among the trypanosomatids, can sensitize Trypanosoma brucei to ganciclovir. Since ganciclovir activity depends upon phosphorylation by ectopically expressed viral thymidine kinase, we also tested the adenosine analog, ara-A, that may be fully phosphorylated by native T. brucei kinase(s). Both Tb927.6.2800 and HD82 knockdowns were resistant to this analog. Tb927.6.2800 knockdown increased sensitivity to hydroxyurea, while dNTP analysis indicated that HD82 is indeed a triphosphohydrolase with dATP as the preferred substrate. Our results provide insights into nucleoside/nucleotide metabolism and nucleoside analog metabolism and resistance in trypanosomatids. We suggest that the product of 6.2800 sensitizes cells to purine analogs through DNA repair, while HD82 does so by reducing the native purine pool.IMPORTANCEThere is substantial interest in developing nucleoside analogs as anti-parasitic agents. We used genome-scale genetic screening and discovered two proteins linked to purine analog resistance in African trypanosomes. Our screens also identified two nucleoside kinases required for pro-drug activation, further validating the approach. The top novel hit, HD82, is related to SAMHD1, a mammalian nuclear viral restriction factor. We validated HD82 and localized the protein to the trypanosome nucleus. HD82 appears to sensitize trypanosomes to nucleoside analogs by reducing native pools of nucleotides, providing insights into both nucleoside/nucleotide metabolism and nucleoside analog resistance in trypanosomatids

HOW SCIENTIFIC PRODUCTION POSITIVELY AFFECT SOCIAL PSYCHOLOGY INTERVENTION? A BIBLIOMETRIC ANALYSIS ON THE SCIENCE OF WELL-BEING

Purpose: The recent decades were witnessed such a rapid rise in wellbeing researches on the field of psychology. Social psychology intervention (SPI) has a strong impact on individual mental health and remarkable achievement in education domain. In this context, subject development within the last years could be illustrated through a bibliometric approach. This research aimed to analyze the scientific productivity while considering global trends of psychological intervention. Methodology: Web of Science Core Collection databases were selected in the period from 1990 to mid-2018. By thoroughly scrutinizing the objects which came to this attention, the trend of documents, publication date, geographic contribution to the field, relevant authorship and most cited articles, as well as frequent keywords from more than thousand references, were investigated. Main Findings: The results clearly indicate that the United States of America dedicates itself with the highest level of attention in the area of social psychology intervention. Whereas, lower attention was focused on the research criteria in Asian continent, except China. Although the majority of the studies on SPI approach is from USA, but performed analysis highlight the vast research potential to validate without any specific considerations to certain region settings or cultures. Implications/Applications: Despite advantages of SPI, the majority of the existed researches has been carried out predominantly in United States. Whereas, from defining the unit of analysis, the hypothesis might worthily be studied by practitioners to generalize the issue over various frameworks

Advantages of the net benefit regression framework for trial-based economic evaluations of cancer treatments: an example from the Canadian Cancer Trials Group CO.17 trial.

BackgroundEconomic evaluations commonly accompany trials of new treatments or interventions; however, regression methods and their corresponding advantages for the analysis of cost-effectiveness data are not widely appreciated.MethodsTo illustrate regression-based economic evaluation, we review a cost-effectiveness analysis conducted by the Canadian Cancer Trials Group's Committee on Economic Analysis and implement net benefit regression.ResultsNet benefit regression offers a simple option for cost-effectiveness analyses of person-level data. By placing economic evaluation in a regression framework, regression-based techniques can facilitate the analysis and provide simple solutions to commonly encountered challenges (e.g., the need to adjust for potential confounders, identify key patient subgroups, and/or summarize "challenging" findings, like when a more effective regimen has the potential to be cost-saving).ConclusionsEconomic evaluations of patient-level data (e.g., from a clinical trial) can use net benefit regression to facilitate analysis and enhance results

Insights into antitrypanosomal drug mode-of-action from cytology-based profiling

Chemotherapy continues to have a major impact on reducing the burden of disease caused by trypanosomatids. Unfortunately though, the mode-of-action (MoA) of antitrypanosomal drugs typically remains unclear or only partially characterised. This is the case for four of five current drugs used to treat Human African Trypanosomiasis (HAT); eflornithine is a specific inhibitor of ornithine decarboxylase. Here, we used a panel of T. brucei cellular assays to probe the MoA of the current HAT drugs. The assays included DNA-staining followed by microscopy and quantitative image analysis, or flow cytometry; terminal dUTP nick end labelling to monitor mitochondrial (kinetoplast) DNA replication; antibody-based detection of sites of nuclear DNA damage; and fluorescent dye-staining of mitochondria or lysosomes. We found that melarsoprol inhibited mitosis; nifurtimox reduced mitochondrial protein abundance; pentamidine triggered progressive loss of kinetoplast DNA and disruption of mitochondrial membrane potential; and suramin inhibited cytokinesis. Thus, current antitrypanosomal drugs perturb distinct and specific cellular compartments, structures or cell cycle phases. Further exploiting the findings, we show that putative mitogen-activated protein-kinases contribute to the melarsoprol-induced mitotic defect, reminiscent of the mitotic arrest associated signalling cascade triggered by arsenicals in mammalian cells, used to treat leukaemia. Thus, cytology-based profiling can rapidly yield novel insight into antitrypanosomal drug MoA

Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids

Trypanosomatids cause the neglected tropical diseases, sleeping sickness, Chagas disease and the leishmaniases. Studies on these lethal parasites would be further facilitated by new and improved genetic technologies. Scalable precision editing methods, for example, could be used to improve our understanding of potential mutations associated with drug resistance, a current priority given that several new anti-trypanosomal drugs, with known targets, are currently in clinical development. We report the development of a simple oligo targeting method for rapid and precise editing of priority drug targets in otherwise wild type trypanosomatids. In Trypanosoma brucei, approx. 50-b single-stranded oligodeoxynucleotides were optimal, multiple base edits could be incorporated, and editing efficiency was substantially increased when mismatch repair was suppressed. Resistance-associated edits were introduced in T. brucei cyclin dependent kinase 12 (CRK12, L(482)F) or cleavage and polyadenylation specificity factor 3 (N(232)H), in the Trypanosoma cruzi proteasome β5 subunit (G(208)S), or in Leishmania donovani CRK12 (G(572)D). We further implemented oligo targeting for site saturation mutagenesis, targeting codon G(492) in T. brucei CRK12. This approach, combined with amplicon sequencing for codon variant scoring, revealed fourteen resistance conferring G(492) edits encoding six distinct amino acids. The outputs confirm on-target drug activity, reveal a variety of resistance-associated mutations, and facilitate rapid assessment of potential impacts on drug efficacy

Trypanosoma brucei BRCA2 acts in a life cycle-specific genome stability process and dictates BRC repeat number-dependent RAD51 subnuclear dynamics

Trypanosoma brucei survives in mammals through antigenic variation, which is driven by RAD51-directed homologous recombination of Variant Surface Glycoproteins (VSG) genes, most of which reside in a subtelomeric repository of >1000 silent genes. A key regulator of RAD51 is BRCA2, which in T. brucei contains a dramatic expansion of a motif that mediates interaction with RAD51, termed the BRC repeats. BRCA2 mutants were made in both tsetse fly-derived and mammal-derived T. brucei, and we show that BRCA2 loss has less impact on the health of the former. In addition, we find that genome instability, a hallmark of BRCA2 loss in other organisms, is only seen in mammal-derived T. brucei. By generating cells expressing BRCA2 variants with altered BRC repeat numbers, we show that the BRC repeat expansion is crucial for RAD51 subnuclear dynamics after DNA damage. Finally, we document surprisingly limited co-localization of BRCA2 and RAD51 in the T. brucei nucleus, and we show that BRCA2 mutants display aberrant cell division, revealing a function distinct from BRC-mediated RAD51 interaction. We propose that BRCA2 acts to maintain the huge VSG repository of T. brucei, and this function has necessitated the evolution of extensive RAD51 interaction via the BRC repeats, allowing re-localization of the recombinase to general genome damage when needed