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The Trypanosoma brucei genome is structurally complex. Eleven megabase-sized chromosomes each comprise a transcribed core flanked by silent subtelomeres, housing thousands of Variant Surface Glycoprotein (VSG) genes. Additionally, hundreds of sub-megabase chromosomes contain 177 bp repeats of unknown function, and VSG transcription sites localise to many telomeres. DNA replication dynamics have only been described in the megabase chromosome cores, and in the single active VSG transcription site. Using a Nanopore genome assembly, we show that megabase chromosome subtelomeres display a paucity of replication initiation events relative to the core, correlating with increased instability. In addition, replication of the active VSG transcription site is shown to originate from the telomere, likely causing targeted VSG recombination. Lastly, we provide evidence that the 177 bp repeats act as conserved DNA replication origins, explaining submegabase chromosome stability. Compartmentalized DNA replication therefore explains how T. brucei balances stable genome transmission with localised instability driving immune evasion., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei , relies on targeted recombination of silent Variant Surface Glycoprotein ( VSG ) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Objective: To examine the prevalence of ADHD and the association of comorbid disorders, and multivariate disorder classes with role impairment in college students., Method: About 15,991 freshmen (24 colleges, 9 countries, WMH-ICS) (response rate = 45.6%) completed online WMH-CIDI-SC surveys for 6-month ADHD and six 12-month DSM-IV disorders. We examined multivariate disorder classes using latent class analysis (LCA) and simulated a population attributable risk proportions (PARPs) of ADHD-related impairment., Results: About 15.9% had ADHD, of which 58.4% had comorbidities. LCA classified ADHD respondents to pure (42.9%), internalizing (36.0%), bipolar comorbidities (11.3%), and externalizing disorder classes (9.8%). ADHD, comorbidities, and multivariate disorder classes independently predicted severe impairment. PARPs: eliminating ADHD hypothetically reduced severe impairment by 19.2%, 10.1% adjusted for comorbidities, 9.5% for multivariate disorder classes., Conclusions: ADHD and comorbid disorders are common and impairing in college students. Personalized transdiagnostic interventions guided by multivariate disorder classes should be explored.

A growing body of evidence supports an important role for alterations in the brain-gut-microbiome axis in the aetiology of depression and other psychiatric disorders. The potential role of the oral microbiome in mental health has received little attention, even though it is one of the most diverse microbiomes in the body and oral dysbiosis has been linked to systemic diseases with an underlying inflammatory aetiology. This study examines the structure and composition of the salivary microbiome for the first time in young adults who met the DSM-IV criteria for depression (n = 40) and matched controls (n = 43) using 16S rRNA gene-based next generation sequencing. Subtle but significant differences in alpha and beta diversity of the salivary microbiome were observed, with clear separation of depressed and healthy control cohorts into distinct clusters. A total of 21 bacterial taxa were found to be differentially abundant in the depressed cohort, including increased Neisseria spp. and Prevotella nigrescens, while 19 taxa had a decreased abundance. In this preliminary study we have shown that the composition of the oral microbiome is associated with depression in young adults. Further studies are now warranted, particuarly investigations into whether such shifts play any role in the underling aetiology of depression., (© 2021. The Author(s).)

DNA replication is needed to duplicate a cell's genome in S phase and segregate it during cell division. Previous work in Leishmania detected DNA replication initiation at just a single region in each chromosome, an organisation predicted to be insufficient for complete genome duplication within S phase. Here, we show that acetylated histone H3 (AcH3), base J and a kinetochore factor co-localise in each chromosome at only a single locus, which corresponds with previously mapped DNA replication initiation regions and is demarcated by localised G/T skew and G4 patterns. In addition, we describe previously undetected subtelomeric DNA replication in G2/M and G1-phase-enriched cells. Finally, we show that subtelomeric DNA replication, unlike chromosome-internal DNA replication, is sensitive to hydroxyurea and dependent on 9-1-1 activity. These findings indicate that Leishmania 's genome duplication programme employs subtelomeric DNA replication initiation, possibly extending beyond S phase, to support predominantly chromosome-internal DNA replication initiation within S phase., Competing Interests: JD, CM, DB, KC, CL, RO, LT, RM No competing interests declared, (© 2020, Damasceno et al.)

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma brucei evades mammalian immunity by using recombination to switch its surface-expressed variant surface glycoprotein (VSG), while ensuring that only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have been implicated in the catalysis of VSG switching by recombination, not transcriptional control. How VSG switching is signaled to guide the appropriate reaction or to integrate switching into parasite growth is unknown. Here, we show that the loss of ATR, a DNA damage-signaling protein kinase, is lethal, causing nuclear genome instability and increased VSG switching through VSG-localized damage. Furthermore, ATR loss leads to the increased transcription of silent VSG expression sites and expression of mixed VSGs on the cell surface, effects that are associated with the altered localization of RNA polymerase I and VEX1. This work shows that ATR acts in antigenic variation both through DNA damage signaling and surface antigen expression control., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ribonucleotides represent a threat to DNA genome stability and transmission. Two types of Ribonuclease H (RNase H) excise ribonucleotides when they form part of the DNA strand, or hydrolyse RNA when it base-pairs with DNA in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast survives the absence of both enzymes. RNase H1 loss is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. Differential gene expression analysis reveals limited overall changes in RNA levels for RNA Pol II genes after TbRH2A loss, but increased perturbation of nucleotide metabolic genes. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, also leading to altered gene expression. Thus, we demonstrate separation of function between two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Early life experiences, such as childhood adversities or poor parenting practices, can impact on the ability to cope with stressors across the lifespan. Furthermore, poor coping skills can lead to the development of mental illnesses, self-harm, and suicidal behavior. This study aimed to examine demographic differences in stress levels and to determine if those who had endured negative childhood experiences would be more likely to develop psychological problems and display suicidal behavior when current stress levels were accounted for. The study also explored the link between coping and mental health problems. Finally, it aimed to predict risk and protective factors related to good coping skills. The study utilized data obtained from the Ulster University Student Wellbeing Study, conducted across four university campuses in Northern Ireland in 2015 (n = 716) as part of the World Health Organization World Mental Health (WMH) International College Student Initiative. Mental health problems and early childhood experiences were examined using questions adapted from the WMH Composite International Diagnostic Interview, with self-harm and suicidal behavior measured using the Self-Injurious Thoughts and Behaviors Interview (SITBI). Females, non-heterosexuals, and older students experienced more current stress. When current stress levels were high, childhood adversities and parental overcontrol and overindulgence were related to higher rates of mental health problems, self-harm, and suicidal behavior. Poor coping skills were associated with negative mental health outcomes. Social support and good emotion-regulation strategies were related to effective coping, while parental overcontrol and overindulgence, female gender, and younger age were related to poorer coping. The study highlights the importance of developing good coping skills to deal with life stressors, thereby minimizing the risk of psychological problems and suicidal behavior. The findings provide support for initiatives to help parents improve their parenting skills and other programs to help young people cope with stress, and to develop social networks and adaptive emotion-regulation strategies., (© 2019 The Institute of Psychology, Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

Background: College students have high prevalence of mental disorders and suicidal thoughts and behaviours, and low rates of treatment uptake. This study assesses treatment access, intentions to seek help, and perceived barriers to help-seeking, considering gender and suicidal thoughts or behaviours (STBs) as predictors., Methods: Data is from the Ulster University Student Wellbeing study (2015) conducted in Northern Ireland (NI), as part of the WHO World Mental Health Surveys International College Student Project. Participants are 392 new college entrants (162 males (41.3%)/230 females (58.7%)), who all reported some lifetime mental disorder or STBs., Results: Receipt of treatment was low (37.8%), particularly among males and those with no STBs. Males were less likely to intend to access external professional services and were less likely than females to rate embarrassment (OR = 0.60) or worry about being treated differently (OR = 0.63) as important reasons for not seeking treatment. Those with STBs rated wanting to handle things on their own as a more important barrier those with no STBs (OR = 0.55 for non STBs group) and rated being unsure where to go as a less important barrier than those with no STBs (OR = 1.80 for non STBs group)., Limitations: Data is correlational and concerns lifetime criteria for mental disorder, with no consideration of current mental status nor disorder type., Conclusions: These findings have implications for the active screening and intervention for vulnerable college students, particularly males and those with mental disorders but no STBs., (Copyright © 2019. Published by Elsevier B.V.)

Background: Although mental disorders and suicidal thoughts-behaviors (suicidal thoughts and behaviors) are common among university students, the majority of students with these problems remain untreated. It is unclear what the barriers are to these students seeking treatment., Aims: The aim of this study is to examine the barriers to future help-seeking and the associations of clinical characteristics with these barriers in a cross-national sample of first-year college students., Method: As part of the World Mental Health International College Student (WMH-ICS) initiative, web-based self-report surveys were obtained from 13,984 first-year students in eight countries across the world. Clinical characteristics examined included screens for common mental disorders and reports about suicidal thoughts and behaviors. Multivariate regression models adjusted for socio-demographic, college-, and treatment-related variables were used to examine correlates of help-seeking intention and barriers to seeking treatment., Results: Only 24.6% of students reported that they would definitely seek treatment if they had a future emotional problem. The most commonly reported reasons not to seek treatment among students who failed to report that they would definitely seek help were the preference to handle the problem alone (56.4%) and wanting to talk with friends or relatives instead (48.0%). Preference to handle the problem alone and feeling too embarrassed were also associated with significantly reduced odds of having at least some intention to seek help among students who failed to report that they would definitely seek help. Having 12-month major depression, alcohol use disorder, and suicidal thoughts and behaviors were also associated with significantly reduced reported odds of the latter outcome., Conclusions: The majority of first-year college students in the WMH-ICS surveys report that they would be hesitant to seek help in case of future emotional problems. Attitudinal barriers and not structural barriers were found to be the most important reported reasons for this hesitation. Experimental research is needed to determine whether intention to seek help and, more importantly, actual help-seeking behavior could be increased with the extent to which intervention strategies need to be tailored to particular student characteristics. Given that the preference to handle problems alone and stigma and appear to be critical, there could be value in determining if internet-based psychological treatments, which can be accessed privately and are often build as self-help approaches, would be more acceptable than other types of treatments to student who report hesitation about seeking treatment., (© 2019 The Authors International Journal of Methods in Psychiatric Research Published by John Wiley & Sons Ltd.)

R-loops are stable RNA-DNA hybrids that have been implicated in transcription initiation and termination, as well as in telomere maintenance, chromatin formation, and genome replication and instability. RNA Polymerase (Pol) II transcription in the protozoan parasite Trypanosoma brucei is highly unusual: virtually all genes are co-transcribed from multigene transcription units, with mRNAs generated by linked trans-splicing and polyadenylation, and transcription initiation sites display no conserved promoter motifs. Here, we describe the genome-wide distribution of R-loops in wild type mammal-infective T. brucei and in mutants lacking RNase H1, revealing both conserved and diverged functions. Conserved localization was found at centromeres, rRNA genes and retrotransposon-associated genes. RNA Pol II transcription initiation sites also displayed R-loops, suggesting a broadly conserved role despite the lack of promoter conservation or transcription initiation regulation. However, the most abundant sites of R-loop enrichment were within the regions between coding sequences of the multigene transcription units, where the hybrids coincide with sites of polyadenylation and nucleosome-depletion. Thus, instead of functioning in transcription termination the most widespread localization of R-loops in T. brucei suggests a novel correlation with pre-mRNA processing. Finally, we find little evidence for correlation between R-loop localization and mapped sites of DNA replication initiation.

Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune evasion strategy that is catalysed both by transcription and recombination reactions, each operating within specialised telomeric VSG expression sites (ES). VSG switching is likely triggered by events focused on the single actively transcribed ES, from a repertoire of around 15, but the nature of such events is unclear. Here we show that RNA-DNA hybrids, called R-loops, form preferentially within sequences termed the 70 bp repeats in the actively transcribed ES, but spread throughout the active and inactive ES, in the absence of RNase H1, which degrades R-loops. Loss of RNase H1 also leads to increased levels of VSG coat switching and replication-associated genome damage, some of which accumulates within the active ES. This work indicates VSG ES architecture elicits R-loop formation, and that these RNA-DNA hybrids connect T. brucei immune evasion by transcription and recombination., Competing Interests: The authors have declared that no competing interests exist.

Background: Prevalence estimates of suicidal behaviour in the college student population are consistently higher than rates for the general adult population. This study examines mental health disorders and childhood adversities as predictors of self-harm and suicidal behaviours., Methods: The Ulster University Student Wellbeing study commenced in September 2015 as part of the WHO World Mental Health Surveys International College Student Project. In Northern Ireland (NI) 739 students participated (462 female, 274 male and 3 other specified), with the WMH-CIDI used to examine psychopathology. Mean age was 21 years old., Results: Thirty-one percent endorsed suicidal ideation (24.3% of males and 36.9% of females) with almost 1 in 5 students having made a plan for suicide in the 12 months prior to the survey. Latent profile analysis revealed three profiles of childhood adversity (high, moderate, and low risk). Logistic regression analyses showed that there was an increased likelihood of all queried self-harm and suicidal behaviours in those who were not heterosexual orientation, and among those with either moderate or high levels of childhood adversities. Probable alcohol dependence was associated with a significantly increased likelihood of suicide attempt or self-harm with either a suicide plan or a suicide attempt., Limitations: Influences of self-report measures and the generalizability of the sample are discussed., Conclusions: Policies and strategies for early identification of those with mental illnesses or adversities that increase their risk, should be prioritised. It would also be useful to identify individuals at risk in secondary schools to allow for additional support to be offered to them during the key time of transitioning into higher education., (Copyright © 2018. Published by Elsevier B.V.)

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating - a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility.

Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue., (© 2011 Blackwell Publishing Ltd.)