The developmental hierarchy and scarcity of replicative slender trypanosomes in blood challenges their role in infection maintenance.

The development of Trypanosoma brucei in its mammalian host is marked by a distinct morphological change as replicative "slender" forms differentiate into cell cycle arrested "stumpy" forms in a quorum-sensing-dependent manner. Although stumpy forms dominate chronic infections at the population level, the proportion of replicative parasites at the individual cell level and the irreversibility of arrest in the bloodstream are unclear. Here, we experimentally demonstrate that developmental cell cycle arrest is definitively irreversible in acute and chronic infections in mice. Furthermore, analysis of replicative capacity and single-cell transcriptome profiling reveal a temporal hierarchy, whereby cell cycle arrest and appearance of a reversible stumpy-like transcriptome precede irreversible commitment and morphological change. Unexpectedly, we show that proliferating parasites are exceptionally scarce in the blood after infections are established. This challenges the ability of bloodstream trypanosomes to sustain infection by proliferation or antigenic variation, these parasites instead being overwhelmingly adapted for transmission.

RAD51-mediated R-loop formation acts to repair transcription-associated DNA breaks driving antigenic variation in Trypanosoma brucei.

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.

Immunoprecipitation of RNA-DNA hybrid interacting proteins in Trypanosoma brucei reveals conserved and novel activities, including in the control of surface antigen expression needed for immune evasion by antigenic variation.

RNA-DNA hybrids are epigenetic features of genomes that provide a diverse and growing range of activities. Understanding of these functions has been informed by characterising the proteins that interact with the hybrids, but all such analyses have so far focused on mammals, meaning it is unclear if a similar spectrum of RNA-DNA hybrid interactors is found in other eukaryotes. The African trypanosome is a single-cell eukaryotic parasite of the Discoba grouping and displays substantial divergence in several aspects of core biology from its mammalian host. Here, we show that DNA-RNA hybrid immunoprecipitation coupled with mass spectrometry recovers 602 putative interactors in T. brucei mammal- and insect-infective cells, some providing activities also found in mammals and some lineage-specific. We demonstrate that loss of three factors, two putative helicases and a RAD51 paralogue, alters T. brucei nuclear RNA-DNA hybrid and DNA damage levels. Moreover, loss of each factor affects the operation of the parasite immune survival mechanism of antigenic variation. Thus, our work reveals the broad range of activities contributed by RNA-DNA hybrids to T. brucei biology, including new functions in host immune evasion as well as activities likely fundamental to eukaryotic genome function.

Read, Write, Adapt: Challenges and Opportunities during Kinetoplastid Genome Replication.

The genomes of all organisms are read throughout their growth and development, generating new copies during cell division and encoding the cellular activities dictated by the genome's content. However, genomes are not invariant information stores but are purposefully altered in minor and major ways, adapting cellular behaviour and driving evolution. Kinetoplastids are eukaryotic microbes that display a wide range of such read-write genome activities, in many cases affecting critical aspects of their biology, such as host adaptation. Here we discuss the range of read-write genome changes found in two well-studied kinetoplastid parasites, Trypanosoma brucei and Leishmania, focusing on recent work that suggests such adaptive genome variation is linked to novel strategies the parasites use to replicate their unconventional genomes.

The Role of Osteocytes in Pre-metastatic Niche Formation.

PURPOSE OF REVIEW: The formation of a pre-metastatic niche (PMN), in which primary cancer cells prime the distant site to be favorable to their engraftment and survival, may help explain the strong osteotropism observed in multiple cancers, such as breast and prostate. PMN formation, which includes extracellular matrix remodeling, increased angiogenesis and vascular permeability, enhanced bone marrow-derived cell recruitment and immune suppression, has mostly been described in soft tissues. In this review, we summarize current literature of PMN formation in bone. We also present evidence of a potential role for osteocytes to be the primary mediators of PMN development. RECENT FINDINGS: Osteocytes regulate the bone microenvironment in myriad ways beyond canonical bone tissue remodeling, including changes that contribute to PMN formation. Perilacunar tissue remodeling, which has been observed in both bone and non-bone metastatic cancers, is a potential mechanism by which osteocyte-cancer cell signaling stimulates changes to the bone microenvironment. Osteocytes also protect against endothelial permeability, including that induced by cancer cells, in a loading-mediated process. Finally, osteocytes are potent regulators of cells within the bone marrow, including progenitors and immune cells, and might be involved in this aspect of PMN formation. Osteocytes should be examined for their role in PMN formation.

Exploring health professionals' knowledge of cancer-related pain: a scoping review.

BACKGROUND: Cancer-related pain is a complex multidimensional experience that affects all aspects of life. To support those impacted by cancer-related pain it is essential that health professionals have adequate knowledge in its assessment and management. AIMS: To explore the knowledge of health professionals regarding cancer-related pain. METHODS: A scoping review methodology was used to systematically search the literature published between 2010 and 2020. Databases CINAHL, Medline and PsycINFO were searched using terms 'cancer pain', 'healthcare professional', 'knowledge', 'attitudes' and 'barriers'. FINDINGS: The search identified 38 articles. They focused on nursing knowledge with minimal involvement of allied health professionals. Knowledge levels were variable with the majority demonstrating poor knowledge. Common knowledge gaps included around fear of addiction, principles of cancer-related pain assessment and management, and interventional techniques. CONCLUSION: Knowledge of cancer-related pain is variable with several knowledge gaps. More work is needed to ensure health professionals have adequate knowledge regarding the complexity of cancer-related pain.

Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation.

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.

Application of single-cell transcriptomics to kinetoplastid research.

Kinetoplastid parasites are responsible for both human and animal diseases across the globe where they have a great impact on health and economic well-being. Many species and life cycle stages are difficult to study due to limitations in isolation and culture, as well as to their existence as heterogeneous populations in hosts and vectors. Single-cell transcriptomics (scRNA-seq) has the capacity to overcome many of these difficulties, and can be leveraged to disentangle heterogeneous populations, highlight genes crucial for propagation through the life cycle, and enable detailed analysis of host­parasite interactions. Here, we provide a review of studies that have applied scRNA-seq to protozoan parasites so far. In addition, we provide an overview of sample preparation and technology choice considerations when planning scRNA-seq experiments, as well as challenges faced when analysing the large amounts of data generated. Finally, we highlight areas of kinetoplastid research that could benefit from scRNA-seq technologies.

Trypanosoma brucei ribonuclease H2A is an essential R-loop processing enzyme whose loss causes DNA damage during transcription initiation and antigenic variation.

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.

Ribonuclease H1-targeted R-loops in surface antigen gene expression sites can direct trypanosome immune evasion.

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.

Evaluation of mechanisms that may generate DNA lesions triggering antigenic variation in African trypanosomes.

Antigenic variation by variant surface glycoprotein (VSG) coat switching in African trypanosomes is one of the most elaborate immune evasion strategies found among pathogens. Changes in the identity of the transcribed VSG gene, which is always flanked by 70-bp and telomeric repeats, can be achieved either by transcriptional or DNA recombination mechanisms. The major route of VSG switching is DNA recombination, which occurs in the bloodstream VSG expression site (ES), a multigenic site transcribed by RNA polymerase I. Recombinogenic VSG switching is frequently catalyzed by homologous recombination (HR), a reaction normally triggered by DNA breaks. However, a clear understanding of how such breaks arise-including whether there is a dedicated and ES-focused mechanism-is lacking. Here, we synthesize data emerging from recent studies that have proposed a range of mechanisms that could generate these breaks: action of a nuclease or nucleases; repetitive DNA, most notably the 70-bp repeats, providing an intra-ES source of instability; DNA breaks derived from the VSG-adjacent telomere; DNA breaks arising from high transcription levels at the active ES; and DNA lesions arising from replication-transcription conflicts in the ES. We discuss the evidence that underpins these switch-initiation models and consider what features and mechanisms might be shared or might allow the models to be tested further. Evaluation of all these models highlights that we still have much to learn about the earliest acting step in VSG switching, which may have the greatest potential for therapeutic intervention in order to undermine the key reaction used by trypanosomes for their survival and propagation in the mammalian host.

Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome.

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.

Genome-wide and protein kinase-focused RNAi screens reveal conserved and novel damage response pathways in Trypanosoma brucei.

All cells are subject to structural damage that must be addressed for continued growth. A wide range of damage affects the genome, meaning multiple pathways have evolved to repair or bypass the resulting DNA lesions. Though many repair pathways are conserved, their presence or function can reflect the life style of individual organisms. To identify genome maintenance pathways in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens to identify genes important for survival following exposure to the alkylating agent methyl methanesulphonate. Amongst a cohort of broadly conserved and, therefore, early evolved repair pathways, we reveal multiple activities not so far examined functionally in T. brucei, including DNA polymerases, DNA helicases and chromatin factors. In addition, the screens reveal Trypanosoma- or kinetoplastid-specific repair-associated activities. We also provide focused analyses of repair-associated protein kinases and show that loss of at least nine, and potentially as many as 30 protein kinases, including a nuclear aurora kinase, sensitises T. brucei to alkylation damage. Our results demonstrate the potential for synthetic lethal genome-wide screening of gene function in T. brucei and provide an evolutionary perspective on the repair pathways that underpin effective responses to damage, with particular relevance for related kinetoplastid pathogens. By revealing that a large number of diverse T. brucei protein kinases act in the response to damage, we expand the range of eukaryotic signalling factors implicated in genome maintenance activities.

Nurses' Experiences of Patients with Substance-Use Disorder in Pain: A Phenomenological Study.

Patients with substance-use disorder and pain are at risk of having their pain underestimated and undertreated. Unrelieved pain can exacerbate characteristics that are believed to be 'drug-seeking' and in turn, perceived drug-seeking behavior can contribute to a patient being stigmatized and labeled 'difficult'. Previous literature has indicated that negative attitudes towards patients with substance-use disorder may affect their pain management but little is known about the specific barriers. This study explored nurses' experiences of working with patients with substance-use disorder in pain, providing an in-depth insight into their perspective. Descriptive phenomenology was employed as a framework for conducting semi-structured interviews to reveal the experiences of registered nurses. A convenience sample of registered nurses from a variety of clinical backgrounds were recruited and interviewed. This rich data was analyzed according to Giorgi's five-stage approach. Participants described feelings of powerlessness and frustration due to patient non-compliance, discrepancies in patient management amongst team members and external pressures effecting pain management. Participants described characteristics believed to be common, including psychosocial factors such as complex social backgrounds or mental health issues. Nurses' education and support needs were identified. Stereotyping and stigmatism were found to potentially still exist, yet there was also a general awareness of some specific clinical issues such as opioid tolerance and opioid-induced hyperalgesia. Further emphasis is required on interprofessional education and communication to improve patient management, alongside an appreciation of patient's rights facilitated by a concordance model of care.

Perceptions and experiences of the subjective well-being of people with glioblastoma: a longitudinal phenomenological study.

Background: Glioblastoma (GBM) is a devastating form of brain cancer, with a short life expectancy. In addition to this poor prognosis, people with GBM often experience symptoms that may have a profound impact on their subjective well-being (SWB). The aim of this study was to investigate the lived experiences and perceptions of people with GBM regarding their SWB. Methods: The study adopted a longitudinal, hermeneutical phenomenological approach. Twenty-seven interviews were conducted with 15 patients over a period of two years. Most participants were interviewed twice on a face-to-face basis (during combined chemotherapy and radiotherapy, and again during adjuvant chemotherapy). The hermeneutic circle was used to guide data analysis. Results: Data analysis identified four key themes that depicted the lived experiences and perceptions of SWB of people with GBM. "Experience of the disease" focuses on the impact of diagnosis, symptoms and side effects. "Daily life" relates to daily activities, family roles, work and social lives. "Coping" includes the importance of normality and goal-setting. "Experiences of care" focuses on the impact of the treatment schedule, experiences of care and impressions of the monitoring of QoL. Conclusion: SWB is affected by a variety of factors throughout the GBM disease and treatment journey. The findings of this study suggest that healthcare professionals can enhance the SWB of people with GBM by providing personalized care that supports people to set themselves goals for the future and retain a degree of normality wherever possible.

Profiling the bloodstream form and procyclic form Trypanosoma brucei cell cycle using single-cell transcriptomics.

African trypanosomes proliferate as bloodstream forms (BSFs) and procyclic forms in the mammal and tsetse fly midgut, respectively. This allows them to colonise the host environment upon infection and ensure life cycle progression. Yet, understanding of the mechanisms that regulate and drive the cell replication cycle of these forms is limited. Using single-cell transcriptomics on unsynchronised cell populations, we have obtained high resolution cell cycle regulated (CCR) transcriptomes of both procyclic and slender BSF Trypanosoma brucei without prior cell sorting or synchronisation. Additionally, we describe an efficient freeze-thawing protocol that allows single-cell transcriptomic analysis of cryopreserved T. brucei. Computational reconstruction of the cell cycle using periodic pseudotime inference allowed the dynamic expression patterns of cycling genes to be profiled for both life cycle forms. Comparative analyses identify a core cycling transcriptome highly conserved between forms, as well as several genes where transcript levels dynamics are form specific. Comparing transcript expression patterns with protein abundance revealed that the majority of genes with periodic cycling transcript and protein levels exhibit a relative delay between peak transcript and protein expression. This work reveals novel detail of the CCR transcriptomes of both forms, which are available for further interrogation via an interactive webtool.

A structured protocol of evidence-based conservative care compared with usual care for acute nonspecific low back pain: a randomized clinical trial.

OBJECTIVE: To compare a protocol of evidence-based conservative care with usual care for acute nonspecific low back pain (LBP) of less than 6 weeks' duration. DESIGN: Parallel-group randomized trial. SETTING: Three practices in the United Kingdom. PARTICIPANTS: Convenience sample of 149 eligible patients were invited to participate in the study, with 118 volunteers being consented and randomly allocated to a treatment group. INTERVENTIONS: The experimental group received evidence-based treatments for acute nonspecific LBP as prescribed in a structured protocol of care developed for this study. The control group received usual conservative care. Participants in both groups could receive up to 7 treatments over a 4-week period. MAIN OUTCOME MEASURES: Oswestry Low Back Disability Index (ODI), visual analog scale (VAS), and Patient Satisfaction Questionnaire, alongside estimation of clinically meaningful outcomes. RESULTS: Total dropout rate was 14% (n=16), with 13% of data missing. Missing data were replaced using a multiple imputation method. Participants in both groups received an average of 6 treatments. There was no statistically significant difference in disability (ODI) scores at the end of week 4 (P=.33), but there was for pain (VAS) scores (P<.001). Interestingly, there were statistically significant differences between the 2 groups for both disability and pain measures at the midpoint of the treatment period (P<.001). Patient satisfaction with care was equally high (85%) in both groups. Minimally clinically important differences in scores and number needed to treat scores (NNT<6) indicated that the experimental treatment (protocol of care) offered a clinically meaningful benefit over the control treatment (usual care), particularly at the midpoint of the treatment period. CONCLUSIONS: Overall, the 2 treatment groups were similar based on primary or secondary outcome measure scores for the full treatment period (4 weeks, with up to 7 treatments). However, there were statistically significant and clinically meaningful differences in both disability and pain scores at week 2 (midpoint) with 4 treatments, suggesting that the protocol of care had a more rapid effect than usual care.

Emergence and adaptation of the cellular machinery directing antigenic variation in the African trypanosome.

Survival of the African trypanosome within its mammalian hosts, and hence transmission between hosts, relies upon antigenic variation, where stochastic changes in the composition of their protective variant-surface glycoprotein (VSG) coat thwart effective removal of the pathogen by adaptive immunity. Antigenic variation has evolved remarkable mechanistic complexity in Trypanosoma brucei, with switching of the VSG coat executed by either transcriptional or recombination reactions. In the former, a single T. brucei cell selectively transcribes one telomeric VSG transcription site, termed the expression site (ES), from a pool of around 15. Silencing of the active ES and activation of one previously silent ES can lead to a co-ordinated VSG coat switch. Outside the ESs, the T. brucei genome contains an enormous archive of silent VSG genes and pseudogenes, which can be recombined into the ES to execute a coat switch. Most such recombination involves gene conversion, including copying of a complete VSG and more complex reactions where novel 'mosaic' VSGs are formed as patchworks of sequences from several silent (pseudo)genes. Understanding of the cellular machinery that directs transcriptional and recombination VSG switching is growing rapidly and the emerging picture is of the use of proteins, complexes and pathways that are not limited to trypanosomes, but are shared across the wider grouping of kinetoplastids and beyond, suggesting co-option of widely used, core cellular reactions. We will review what is known about the machinery of antigenic variation and discuss if there remains the possibility of trypanosome adaptations, or even trypanosome-specific machineries, that might offer opportunities to impair this crucial parasite-survival process.

Machine Learning for Risk Prediction of Oesophago-Gastric Cancer in Primary Care: Comparison with Existing Risk-Assessment Tools.

Oesophago-gastric cancer is difficult to diagnose in the early stages given its typical non-specific initial manifestation. We hypothesise that machine learning can improve upon the diagnostic performance of current primary care risk-assessment tools by using advanced analytical techniques to exploit the wealth of evidence available in the electronic health record. We used a primary care electronic health record dataset derived from the UK General Practice Research Database (7471 cases; 32,877 controls) and developed five probabilistic machine learning classifiers: Support Vector Machine, Random Forest, Logistic Regression, Naïve Bayes, and Extreme Gradient Boosted Decision Trees. Features included basic demographics, symptoms, and lab test results. The Logistic Regression, Support Vector Machine, and Extreme Gradient Boosted Decision Tree models achieved the highest performance in terms of accuracy and AUROC (0.89 accuracy, 0.87 AUROC), outperforming a current UK oesophago-gastric cancer risk-assessment tool (ogRAT). Machine learning also identified more cancer patients than the ogRAT: 11.0% more with little to no effect on false positives, or up to 25.0% more with a slight increase in false positives (for Logistic Regression, results threshold-dependent). Feature contribution estimates and individual prediction explanations indicated clinical relevance. We conclude that machine learning could improve primary care cancer risk-assessment tools, potentially helping clinicians to identify additional cancer cases earlier. This could, in turn, improve survival outcomes.

Paving the Way: Contributions of Big Data to Apicomplexan and Kinetoplastid Research.

In the age of big data an important question is how to ensure we make the most out of the resources we generate. In this review, we discuss the major methods used in Apicomplexan and Kinetoplastid research to produce big datasets and advance our understanding of Plasmodium, Toxoplasma, Cryptosporidium, Trypanosoma and Leishmania biology. We debate the benefits and limitations of the current technologies, and propose future advancements that may be key to improving our use of these techniques. Finally, we consider the difficulties the field faces when trying to make the most of the abundance of data that has already been, and will continue to be, generated.