Attitudes of cardiac rehabilitation and stroke teams towards integration of stroke survivors into adapted cardiac rehabilitation: A focus group study.

OBJECTIVE: To explore the attitudes and beliefs of cardiac rehabilitation and stroke teams towards adapted cardiac rehabilitation, and the broader topics of exercise, healthy lifestyles and health behaviour change, for people with mild-to-moderate severity stroke in the sub-acute phase of recovery. DESIGN: Qualitative focus group-based study. SETTING: Acute and community national health service trusts. PARTICIPANTS: Stroke and cardiac rehabilitation team members. INTERVENTION: Adapted cardiac rehabilitation. MAIN MEASURES: Focus groups. Thematic analysis was applied to the transcribed data. RESULTS: Overall, 57 health professionals participated in 12 focus groups. Positive impacts for teams and stroke survivors were identified particularly confidence. However, there were negatives, barriers and adaptations identified. In addition, there was a lack of knowledge for cardiac rehabilitation teams in relation to stroke survivors and stroke teams in relation to cardiac rehabilitation, exercise and healthy lifestyles. CONCLUSIONS: Cardiac rehabilitation and stroke staff attitudes to cardiac rehabilitation for stroke survivors showed a range of benefits, negatives, barriers and adaptations needed. Confidence and knowledge of the cardiac rehabilitation and stroke teams needs to be addressed. REGISTRATION: ISRCTN65957980.

Evolutionary analysis of cellular reduction and anaerobicity in the hyper-prevalent gut microbe Blastocystis.

Blastocystis is the most prevalent microbial eukaryote in the human and animal gut, yet its role as commensal or parasite is still under debate. Blastocystis has clearly undergone evolutionary adaptation to the gut environment and possesses minimal cellular compartmentalization, reduced anaerobic mitochondria, no flagella, and no reported peroxisomes. To address this poorly understood evolutionary transition, we have taken a multi-disciplinary approach to characterize Proteromonas lacertae, the closest canonical stramenopile relative of Blastocystis. Genomic data reveal an abundance of unique genes in P. lacertae but also reductive evolution of the genomic complement in Blastocystis. Comparative genomic analysis sheds light on flagellar evolution, including 37 new candidate components implicated with mastigonemes, the stramenopile morphological hallmark. The P. lacertae membrane-trafficking system (MTS) complement is only slightly more canonical than that of Blastocystis, but notably, we identified that both organisms encode the complete enigmatic endocytic TSET complex, a first for the entire stramenopile lineage. Investigation also details the modulation of mitochondrial composition and metabolism in both P. lacertae and Blastocystis. Unexpectedly, we identify in P. lacertae the most reduced peroxisome-derived organelle reported to date, which leads us to speculate on a mechanism of constraint guiding the dynamics of peroxisome-mitochondrion reductive evolution on the path to anaerobiosis. Overall, these analyses provide a launching point to investigate organellar evolution and reveal in detail the evolutionary path that Blastocystis has taken from a canonical flagellated protist to the hyper-divergent and hyper-prevalent animal and human gut microbe.

An international survey of experiences and attitudes towards pacing using a heart rate monitor for people with myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: Myalgic encephalomyelitis (ME) is a complex, multi-system neurological condition. The defining feature of ME is post-exertional malaise (PEM) with over 30 symptoms triggered by physical, cognitive, emotional and social activity. The cause of PEM is unclear but one area of research using cardio-pulmonary exercise tests show a reduced ventilatory anaerobic threshold (VAT) with repeated tests leading to PEM. Pacing with heart rate monitoring (HRM) provides feedback to maintain activity intensity below the VAT. There is only one piece of research investigating the use of HRM although a number of guidelines recommend it. OBJECTIVE: To identify the experiences and attitudes of people with ME towards HRM. METHODS: A 40 question online survey was devised and released on ME websites, Twitter and Facebook pages. People with ME read the information sheet and followed an online link to the survey. The survey was open for three weeks and all answers were anonymous. RESULTS: 488 people with ME completed the survey. Most participants were female, 35-50 years and with a reported illness of greater than 5 years. Over 100 types of HR monitor used. Over 30 benefits and over 30 negatives identified. HRM reduced severity of ME and severity and duration of PEM. CONCLUSION: Although there are limitations, HRM has many benefits including helping PwME to understand and manage their PEM and support them to increase their activities, including work. There is a need for more research and education of healthcare professionals in the safe use of HRM.

Repositioning of a Diaminothiazole Series Confirmed to Target the Cyclin-Dependent Kinase CRK12 for Use in the Treatment of African Animal Trypanosomiasis.

African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites Trypanosoma congolense and Trypanosoma vivax, is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both T. congolense and T. vivax via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12.

Evaluating the impact of hydropower on downstream migrating anguillid eels: Catchment-wide and fine-scale approaches to identify cost-effective solutions.

Hydropower is an increasingly popular source of renewable and 'green' (in terms of emissions) energy, but reduced longitudinal connectivity and diverting flow through turbines can have negative impacts on catadromous anguillid eel species that have declined globally. There is an urgent need for environmental managers to perform remediation actions, such as protecting flows for migratory fish and providing passage solutions at infrastructure, under increasing legislative pressure. To deliver this, a more comprehensive understanding of eel migration in catchments with hydropower is required. Here, we illustrate the importance of catchment-wide and fine-scale acoustic telemetry, coupled with the influence of eel maturation (i.e. sex steroid levels), to determine the impact of Wairua run-of-river Power Station (WPS) on downstream migrating shortfin eels (Anguilla australis; n = 25) in Wairua River, New Zealand. Migration speed through the unregulated reach upstream of WPS was positively correlated with flow, but not eel length or sex steroids. Three eels passed a diversion weir (DW) to follow the natural watercourse and eight entered the WPS canal. Eels predominantly entered (95.2%) and were last detected (85.7%) in WPS forebay during hours of darkness. Eleven (52%) of the 21 eels that entered WPS forebay were impinged or entrained, all when three or four turbines were in operation (power generation >3.04 MW). Ten (48%) passed WPS spillway during significantly higher spill than impinged or entrained eels, with four passing during no turbine operation, after experiencing high flows near the intake (multiple receivers in WPS forebay used to quantify fine-scale behaviour). On average, eels were impinged or entrained at WPS significantly quicker (6.40 ± 11.13 days) than eels that entered the spillway (25.17 ± 15.12 days), but eel length and sex steroids did not significantly influence fate. Of the eels that migrated through the entire 55 km study reach, passage time at DW and WPS equated to 0.01-0.02% and 47.62-92.17% of their migration, respectively. Mitigation for WPS (and similar power schemes) should focus on operational or physical changes at DW to minimise eels entering power station forebay(s). Turbine shutdowns, ensuring WPS spillway is available and the provision of a bypass channel in WPS forebay are also discussed as ways to conserve the species with the potential to save costs for water resource managers.

Does cardiac rehabilitation for people with stroke in the sub-acute phase of recovery lead to physical behaviour change? Results from compositional analysis of accelerometry-derived data.

OBJECTIVES: Does adapted cardiac rehabilitation (CR) improve the physical behaviours of people with mild-to-moderate stroke in the sub-acute recovery phase using a compositional data analysis (CoDA) approach? DESIGN: Before-after. SETTING: University Hospitals of Leicester, Glenfield Hospital, UK. PARTICIPANTS: 24 individuals completed CR and provided valid physical activity (PA) data (mean (SD) 63.1 (14.6) years, 58% male (14/24)). INTERVENTION: 6-week adapted CR program within 6-months of stroke. MAIN OUTCOME MEASURES: Physical behaviours were assessed using waist-worn accelerometry. Step count, stationary time (ST), light PA (LPA), and moderate-to-vigorous PA (MVPA) were compared pre post CR using conventional analyses and CoDA. Analysed compositions were: Waking day (ST, LPA, MVPA); ST (1-9-minutes, 10-29-minutes, ≥30-minutes bouts); and MVPA (1-4-minutes, 5-9-minutes, ≥10-minutes bouts). RESULTS: Following CR, patients took significantly more steps (mean (SD) 3255 (2864) vs 3908 (3399) steps/day, P=0.004) and engaged in more bouts of MVPA lasting ≥5 and ≥10-minutes (≥5-minutes: mean (SD) 0.7 (1.4) vs 1.2 (1.8) bouts/day, P=0.008). Using CoDA, no changes in waking day or ST compositions occurred. For waking day, 42% (10/24) increased their LPA and MVPA at the expense of ST. For ST, 33% (8/24) increased their short bouts at the expense of medium and long bouts. For MVPA, 13% (3/24) increased their medium and long bouts at the expense of short bouts. CONCLUSION: People with stroke in the sub-acute stage of recovery exhibited low levels of PA. CR appears to be an effective intervention to increase step count but did not alter the overall proportion of time individuals spent being sedentary, or engaging in LPA or in MVPA. REGISTRATION: ISRCTN65957980.

Chemogenomic Profiling of Antileishmanial Efficacy and Resistance in the Related Kinetoplastid Parasite Trypanosoma brucei.

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative ß-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.

The validity and reliability of the Incremental Shuttle Walk Test and Six-minute Walk Test compared to an Incremental Cycle Test for people who have had a mild-to-moderate stroke.

OBJECTIVE: To determine the construct validity and test re-test reliability of the Six-minute Walk Test (6MWT) and Incremental Shuttle Walk Test (ISWT) in the sub-acute recovery phase following mild-to-moderate severity stroke. PARTICIPANTS: 40 stroke patients (mean age: 68.27 years, SD: 13.48) of median National Institutes of Health Stroke Scale (NIHSS) score 1.2 (range: 0 to 8) within six months of stroke. METHOD: Each participant completed one Incremental Cycle Test (ICT) followed by two ISWT and two 6MWT in a randomised order. Pearson's Correlation Coefficients were used to determine the validity and Bland Altman plots were used to determine the test re-test reliability. RESULTS: The Incremental Cycle Test (ICT) was positively correlated with the ISWT (r=0.59, 95% confidence intervals 0.35 to 0.76, P=0.001) and the 6MWT (0.55, 0.35 to 0.71, P<0.001). The correlation of the ICT with the ISWT and 6MWT was higher for the 17 patients with no residual (ISWT: r=0.79, P<0.001; 6MWT: 0.826, P<0.001) compared to mild-to-moderate neurological impairment (ISWT: r=0.45, P=0.03; 6MWT: r=0.38, P=0.08). Test-retest reliability for both the ISWT and the 6MWT showed that there was some variability between the first and second tests with a better performance on the second test. CONCLUSION: The ISWT and 6MWT have a significant, modest correlation with the ICT for stroke patients in the sub-acute recovery phase. The ISWT and 6MWT are not strongly correlated with ICT (VO2 peak) in a stroke population that is disabled. The test-retest reliability of the ISWT and 6MWT indicated that two tests may be needed to accurately assess an individual's capabilities.

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.

Can emergency physicians accurately distinguish retinal detachment from posterior vitreous detachment with point-of-care ocular ultrasound?

STUDY OBJECTIVE: There is significant overlap between the symptoms of patients presenting with retinal detachment (RD) and posterior vitreous detachment (PVD). Urgency to obtain consultation and treatment are dependent on the ability to accurately distinguish these two conditions. The objective of this study was to determine the ability of emergency physicians to differentiate RDs from PVDs using point-of-care (POC) ocular ultrasound. METHODS: Single blinded cross-sectional study at an academic medical center. Emergency physicians with varying ultrasound experience completed a brief tutorial on the sonographic findings of RD and PVD. Thirty POC ocular ultrasound clips obtained from ED patients with ocular symptoms were presented to emergency physicians. The sonographic findings in these clips were in agreement with the final diagnosis made by consultant ophthalmologists. There were 14 ultrasound videos showing PVD, 13 videos showing RD, and 3 normal ocular ultrasound videos. The subjects independently reviewed POC ocular ultrasound video clips and submitted their final interpretations. RESULTS: A total of 390 ocular video clips were reviewed by 13 emergency physicians. Overall, physicians were able to accurately diagnose the presence of a RD 74.6% (95%CI, 69.8-79.4) of the time, PVD 85.7% (95%CI, 77.6-93.8) of the time, and normal ultrasounds 94.9% (95%CI 87.3-100.0) of the time. There was no statistically significant relationship between correct diagnoses for ocular abnormalities or normal ultrasound images and number of previous ocular ultrasounds performed by emergency physicians. CONCLUSION: Emergency physicians were modestly accurate in distinguishing RD from PVD on POC ultrasound.

Emergency department diagnosis of an ovarian inguinal hernia in an 11-year-old female using pointof-care ultrasound / 世界急诊医学杂志（英文）

@#Inguinal hernias affect 5% of children and are usually defined as a protrusion of intestine or omentum through abdominal wall or inguinal canal defects.[1] Inguinal hernias may contain structures other than bowel and unique cases have been documented since the early 1900's.[2–10] Ultrasound has been demonstrated to differentiate superficial swellings and has been used by radiologists to evaluate inguinal masses for decades.[1–5,11–13] Although the use of radiology-performed ultrasound for the diagnosis of congeni tal inguinal hernias containing ovaries, uterus, and fallopian tubes has been documented; the use of point-of-care ultrasound for the evaluation of the acute inguinal mass prior to reduction has not been demonstrated. Accurate identification by the emergency physician of the herniated structures may lead to earlier diagnosis, faster consultation, improve patient management, and superior patient outcomes.

Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity.

Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for melarsoprol and pentamidine-uptake, and melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to melarsoprol and respiratory inhibitors described here might be exploited.

Point-of-care echocardiography in simulation-based education and assessment.

BACKGROUND: Emergency medicine milestones released by the Accreditation Council for Graduate Medical Education require residents to demonstrate competency in bedside ultrasound (US). The acquisition of these skills necessitates a combination of exposure to clinical pathology, hands-on US training, and feedback. OBJECTIVES: We describe a novel simulation-based educational and assessment tool designed to evaluate emergency medicine residents' competency in point-of-care echocardiography for evaluation of a hypotensive patient with chest pain using bedside US. METHODS: This was a cross-sectional study conducted at an academic medical center. A simulation-based module was developed to teach and assess the use of point-of-care echocardiography in the evaluation of the hypotensive patient. The focus of this module was sonographic imaging of cardiac pathology, and this focus was incorporated in all components of the session: asynchronous learning, didactic lecture, case-based learning, and hands-on stations. RESULTS: A total of 52 residents with varying US experience participated in this study. Questions focused on knowledge assessment demonstrated improvement across the postgraduate year (PGY) of training. Objective standardized clinical examination evaluation demonstrated improvement between PGY I and PGY III; however, it was noted that there was a small dip in hands-on scanning skills during the PGY II. Clinical diagnosis and management skills also demonstrated incremental improvement across the PGY of training. CONCLUSION: The 1-day, simulation-based US workshop was an effective educational and assessment tool at our institution.

Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense.

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine.

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2.

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.

Sonography and hypotension: a change to critical problem solving in undergraduate medical education.

STUDY OBJECTIVES: Multiple curricula have been designed to teach medical students the basics of ultrasound; however, few focus on critical problem-solving. The objective of this study is to determine whether a theme-based ultrasound teaching session, dedicated to the use of ultrasound in the management of the hypotensive patient, can impact medical students' ultrasound education and provide critical problem-solving exercises. METHODS: This was a cross-sectional study using an innovative approach to train 3rd year medical students during a 1-day ultrasound training session. The students received a 1-hour didactic session on basic ultrasound physics and knobology and were also provided with YouTube hyperlinks, and links to smart phone educational applications, which demonstrated a variety of bedside ultrasound techniques. In small group sessions, students learned how to evaluate patients for pathology associated with hypotension. A knowledge assessment questionnaire was administered at the end of the session and again 3 months later. Student knowledge was also assessed using different clinical scenarios with multiple-choice questions. RESULTS: One hundred and three 3rd year medical students participated in this study. Appropriate type of ultrasound was selected and accurate diagnosis was made in different hypotension clinical scenarios: pulmonary embolism, 81% (95% CI, 73%-89%); abdominal aortic aneurysm, 100%; and pneumothorax, 89% (95% CI, 82%-95%). The average confidence level in performing ultrasound-guided central line placement was 7/10, focused assessment with sonography for trauma was 8/10, inferior vena cava assessment was 8/10, evaluation for abdominal aortic aneurysm was 8/10, assessment for deep vein thrombus was 8/10, and cardiac ultrasound for contractility and overall function was 7/10. Student performance in the knowledge assessment portion of the questionnaire was an average of 74% (SD =11%) at the end of workshop and 74% (SD =12%) 3 months later (P=0.00). CONCLUSION: At our institution, we successfully integrated ultrasound and critical problem-solving instruction, as part of a 1-day workshop for undergraduate medical education.

Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes.

Kinetoplastid parasites cause lethal diseases in humans and animals. The kinetoplast itself contains the mitochondrial genome, comprising a huge, complex DNA network that is also an important drug target. Isometamidium, for example, is a key veterinary drug that accumulates in the kinetoplast in African trypanosomes. Kinetoplast independence and isometamidium resistance are observed where certain mutations in the F1-γ-subunit of the two-sector F1Fo-ATP synthase allow for Fo-independent generation of a mitochondrial membrane potential. To further explore kinetoplast biology and drug resistance, we screened a genome-scale RNA interference library in African trypanosomes for isometamidium resistance mechanisms. Our screen identified 14 V-ATPase subunits and all 4 adaptin-3 subunits, implicating acidic compartment defects in resistance; V-ATPase acidifies lysosomes and related organelles, whereas adaptin-3 is responsible for trafficking among these organelles. Independent strains with depleted V-ATPase or adaptin-3 subunits were isometamidium resistant, and chemical inhibition of the V-ATPase phenocopied this effect. While drug accumulation in the kinetoplast continued after V-ATPase subunit depletion, acriflavine-induced kinetoplast loss was specifically tolerated in these cells and in cells depleted for adaptin-3 or endoplasmic reticulum membrane complex subunits, also identified in our screen. Consistent with kinetoplast dispensability, V-ATPase defective cells were oligomycin resistant, suggesting ATP synthase uncoupling and bypass of the normal Fo-A6-subunit requirement; this subunit is the only kinetoplast-encoded product ultimately required for viability in bloodstream-form trypanosomes. Thus, we describe 30 genes and 3 protein complexes associated with kinetoplast-dependent growth. Mutations affecting these genes could explain natural cases of dyskinetoplasty and multidrug resistance. Our results also reveal potentially conserved communication between the compartmentalized two-sector rotary ATPases.

Chimerization at the AQP2-AQP3 locus is the genetic basis of melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates.

Aquaglyceroporin-2 is a known determinant of melarsoprol-pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2-AQP3 tandem locus was described from melarsoprol-pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2-aqp3 null T. b. brucei does not. This proves that AQP2-AQP3 chimerization is the cause of melarsoprol-pentamidine cross-resistance in the T. b. gambiense isolates.