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African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism., Protein kinases are fundamental in cellular signalling required for Leishmania survival throughout the life cycle. Here, Baker and Catta-Preta et al. report on a kinome-wide functional study in Leishmania mexicana to define protein kinases with roles in life cycle transition.

We aimed to increase smoking cessation referrals by 30% within three months, to improve identification of patients’ smoking status, offer patients very brief advice (VBA) and increase the prescription of nicotine replacement therapy (NRT). Our multidisciplinary team (MDT) used ‘plan-do-see-act’ methodology to conduct a quality improvement project to increase referrals to smoking cessation services in a busy acute cardio-respiratory admission unit (CDU). We collected baseline and fortnightly data for 17 weeks documenting smoking status (%), VBA (%), NRT prescription (%) and referrals to smoking cessation services (%). The interventions were ‘smoking cessation’ champions, MDT education, increased availability of NRT on CDU with an ‘opt-out’ approach and a documentation ‘sticker’ for the medical records. We compared NRT prescription with the continuous Royal College of Physicians national COPD audit data. 278 medical notes were reviewed between 2/11/2018 to 16/03/2019. Referrals to smoking cessation services from CDU increased from 20% to 50% after the interventions were implemented. Documentation of smoking status ranged from 80% to 100%. VBA and NRT prescription increased from an average of 15% to a peak of 100% but subsequently decreased to 25 and 23%, respectively. Our COPD national audit results show that for patients with COPD, NRT prescription increased during the intervention period and was higher than the national average. We exceeded our goal by at least doubling the number of smoking cessation referrals with minimal resources by forming a multi-disciplinary team of existing healthcare professionals to prioritise ‘smoking cessation’ on a busy acute unit.

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.

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., 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 β-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 design, synthesis and structure-activity relationships of a series of oxazole-benzamide inhibitors of the essential bacterial cell division protein FtsZ are described. Compounds had potent anti-staphylococcal activity and inhibited the cytokinesis of the clinically-significant bacterial pathogen Staphylococcus aureus. Selected analogues possessing a 5-halo oxazole also inhibited a strain of S. aureus harbouring the glycine-to-alanine amino acid substitution at residue 196 of FtsZ which conferred resistance to previously reported inhibitors in the series. Substitutions to the pseudo-benzylic carbon of the scaffold improved the pharmacokinetic properties by increasing metabolic stability and provided a mechanism for creating pro-drugs. Combining multiple substitutions based on the findings reported in this study has provided small-molecule inhibitors of FtsZ with enhanced in vitro and in vivo antibacterial efficacy.

SUMMARYThe trypanosomes cause two neglected tropical diseases, Chagas disease in the Americas and African trypanosomiasis in sub-Saharan Africa. Over recent years a raft of molecular tools have been developed enabling the genetic dissection of many aspects of trypanosome biology, including the mechanisms underlying resistance to some of the current clinical and veterinary drugs. This has led to the identification and characterization of key resistance determinants, including transporters for the anti-Trypanosoma bruceidrugs, melarsoprol, pentamidine and eflornithine, and the activator of nifurtimox-benznidazole, the anti-Trypanosoma cruzidrugs. More recently, advances in sequencing technology, combined with the development of RNA interference libraries in the clinically relevant bloodstream form ofT. bruceihave led to an exponential increase in the number of proteins known to interact either directly or indirectly with the anti-trypanosomal drugs. In this review, we discuss these findings and the technological developments that are set to further revolutionise our understanding of drug-trypanosome interactions. The new knowledge gained should inform the development of novel interventions against the devastating diseases caused by these parasites.

The bacterial cell division protein FtsZ is an attractive target for small-molecule antibacterial drug discovery. Derivatives of 3-methoxybenzamide, including compound PC190723, have been reported to be potent and selective antistaphylococcal agents which exert their effects through the disruption of intracellular FtsZ function. Here, we report the further optimization of 3-methoxybenzamide derivatives towards a drug candidate. The in vitro and in vivo characterization of a more advanced lead compound, designated compound 1, is described. Compound 1 was potently antibacterial, with an average MIC of 0.12 μg/ml against all staphylococcal species, including methicillin- and multidrug-resistant Staphylococcus aureus and Staphylococcus epidermidis . Compound 1 inhibited an S. aureus strain carrying the G196A mutation in FtsZ, which confers resistance to PC190723. Like PC190723, compound 1 acted on whole bacterial cells by blocking cytokinesis. No interactions between compound 1 and a diverse panel of antibiotics were measured in checkerboard experiments. Compound 1 displayed suitable in vitro pharmaceutical properties and a favorable in vivo pharmacokinetic profile following intravenous and oral administration, with a calculated bioavailability of 82.0% in mice. Compound 1 demonstrated efficacy in a murine model of systemic S. aureus infection and caused a significant decrease in the bacterial load in the thigh infection model. A greater reduction in the number of S. aureus cells recovered from infected thighs, equivalent to 3.68 log units, than in those recovered from controls was achieved using a succinate prodrug of compound 1, which was designated compound 2. In summary, optimized derivatives of 3-methoxybenzamide may yield a first-in-class FtsZ inhibitor for the treatment of antibiotic-resistant staphylococcal infections.

Richard Amini,Lori A Stolz,Parisa P Javedani,Kevin Gaskin,Nicola Baker,Vivienne Ng,Srikar Adhikari Department of Emergency Medicine, University of Arizona Medical Center, Tucson, AZ, USA 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. Keywords: point-of care ultrasound, simulation education

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-Leu131 in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine. Electronic supplementary material The online version of this article (doi:10.1007/s00018-016-2173-6) contains supplementary material, which is available to authorized users.

Richard Amini, Lori A Stolz, Nicholas C Hernandez, Kevin Gaskin, Nicola Baker, Arthur Barry Sanders, Srikar AdhikariDepartment of Emergency Medicine, University of Arizona Medical Center, College of Medicine, University of Arizona, Tucson, AZ, USAStudy 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.Keywords: point-of-care ultrasound, undergraduate medical education, protocol-driven education, problem-based learning

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., Author Summary Pentamidine is usable only in the early, non-cerebral stage of human African trypanosomiasis. An aquaglyceroporin, TbAQP2, is required for high-affinity pentamidine uptake. This study established the mechanism. Pentamidine does not pass TbAQP2 but blocks the unique selectivity filter via a single charged amidine and displacement of water from the channel, rendering it the first non-covalent, nanomolar aquaglyceroporin inhibitor. TbAQP2 does not act as a pentamidine transporter but a high-affinity receptor indicating drug uptake by endocytosis. The novel binding mode and proposed uptake mechanism open up possibilities for designing needed anti-trypanosomal small molecule drugs with regard to penetrating the blood-brain barrier for treatment of the severe, second disease stage.

Highlights • Expression of AQP2 restores drug susceptibility in a resistant Trypanosoma brucei gambiense isolate. • The AQP2/3 chimera from the resistant isolate does not complement AQP2 deletion. • Hence AQP2/3 chimerization accompanied by loss of AQP2 is the cause of drug resistance., Graphical Abstract, 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.

International audience; The ability to simultaneously assess every gene in a genome for a role in a particular process has obvious appeal. This protocol describes how to perform genome-scale RNAi library screens in bloodstream-form African trypanosomes, a family of parasites that causes lethal human and animal diseases and also serves as a model for studies on basic aspects of eukaryotic biology and evolution. We discuss strain assembly, screen design and implementation, the RNAi target sequencing approach and hit validation, and we provide a step-by-step protocol. A screen can yield from one to thousands of 'hits' associated with the phenotype of interest. The screening protocol itself takes 2 weeks or less to be completed, and high-throughput sequencing may also be completed within weeks. Pre- and post-screen strain assembly, validation and follow-up can take several months, depending on the type of screen and the number of hits analyzed.

Radiologic studies are a vital component in the workup and diagnosis of disease. Although an indispensable adjunct, they should not be used as a substitute for a thorough history and physical examination. Multiple imaging modalities are available for use. Each has its own advantages, disadvantages, and safety profile, and practitioners should be versed in these when selecting how to work up disease. An appropriate radiographic study will accurately rule in or rule out disease with the least possible harm. Harm is often thought of as pain or physical injury, but broader definitions can include time, cost, radiation, and psychological stress to the patient. When compared with outpatient diagnostics, imaging choices within the emergency setting favor sensitivity over specificity to rule out diseases that require emergent intervention. A stepwise radiological approach may be used to identify illness. Accordingly, the time, cost, and potential harm of imaging studies may increase over time as a specific diagnosis is pursued. Special considerations are necessary for the imaging of children. Sedation or anxiolysis may be required for either computed tomography (CT) or magnetic resonance imaging (MRI). The pediatric emergency provider should be trained in techniques for

Melarsoprol and pentamidine represent the two main classes of drugs, the arsenicals and diamidines, historically used to treat the diseases caused by African trypanosomes: sleeping sickness in humans and Nagana in livestock. Cross-resistance to these drugs was first observed over sixty years ago and remains the only example of cross-resistance among sleeping sickness therapies. A Trypanosoma brucei adenosine transporter is well-known for its role in the uptake of both drugs. More recently, aquaglyceroporin 2 (AQP2) loss-of-function was linked to melarsoprol-pentamidine cross-resistance. AQP2, a channel that appears to facilitate drug accumulation, may also be linked to clinical cases of resistance. Here, we review these findings and consider some new questions as well as future prospects for tackling the devastating diseases caused by these parasites.

African trypanosomes cause sleeping sickness in humans, a disease that is typically fatal without chemotherapy. Unfortunately, drug resistance is common and melarsoprol-resistant trypanosomes often display cross-resistance to pentamidine. Although melarsoprol/pentamidine cross-resistance (MPXR) has been an area of intense interest for several decades, our understanding of the underlying mechanisms remains incomplete. Recently, a locus encoding two closely related aquaglyceroporins, AQP2 and AQP3, was linked to MPXR in a high-throughput loss-of-function screen. Here, we show that AQP2 has an unconventional “selectivity filter.” AQP2 -specific gene knockout generated MPXR trypanosomes but did not affect resistance to a lipophilic arsenical, whereas recombinant AQP2 reversed MPXR in cells lacking native AQP2 and AQP3 . AQP2 was also shown to be disrupted in a laboratory-selected MPXR strain. Both AQP2 and AQP3 gained access to the surface plasma membrane in insect life-cycle–stage trypanosomes but, remarkably, AQP2 was specifically restricted to the flagellar pocket in the bloodstream stage. We conclude that the unconventional aquaglyceroporin, AQP2, renders cells sensitive to both melarsoprol and pentamidine and that loss of AQP2 function could explain cases of innate and acquired MPXR.

Introduction Early goal-directed therapy increases survival in persons with sepsis but requires placement of a central line. We evaluate alternative methods to measuring central venous pressure (CVP) to assess volume status, including peripheral venous pressure (PVP) and stroke volume variation (SVV), which may facilitate nurse-driven resuscitation protocols. Methods Patients were enrolled in the emergency department or ICU of an academic medical center. Measurements of CVP, PVP, SVV, shoulder and elbow position, and dichotomous variables Awake, Movement, and Vented were measured and recorded 7 times during a 1-hour period. Regression analysis was used to predict CVP from PVP and/or SVV, shoulder/elbow position, and dichotomous variables. Results Twenty patients were enrolled, of which 20 had PVP measurements and 11 also had SVV measurements. Multiple regression analysis demonstrated significant predictive relationships for CVP using PVP (CVP = 6.7701 + 0.2312 × PVP – 0.1288 × Shoulder + 12.127 × Movement – 4.4805 × Neck line), SVV (CVP = 14.578 – 0.3951 × SVV + 18.113 × Movement), and SVV and PVP (CVP = 4.2997 – 1.1675 × SVV + 0.3866 × PVP + 18.246 × Awake + 0.1467 × Shoulder=0.4525 × Elbow + 15.472 × Foot line + 10.202 × Arm line). Discussion PVP and SVV are moderately good predictors of CVP. Combining PVP and SVV and adding variables related to body position, movement, ventilation, and sleep/wake state further improves the predictive value of the model. The models illustrate the importance of standardizing patient position, minimizing movement, and placing intravenous lines proximally in the upper extremity or neck.

Summary The concept of specific chemotherapy was developed a century ago by Paul Ehrlich and others. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work 1,2, and the drugs that emerged remain in use for treating Human African Trypanosomiasis (HAT) 3. Ehrlich recognised the importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies, but these mechanisms have remained largely mysterious. Here, we use all five current HAT drugs for genome-scale RNA interference (RNAi) target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling identifies both known drug importers 4,5 and the only known pro-drug activator 6, and links more than fifty additional genes to drug action. A specific bloodstream stage invariant surface glycoprotein (ISG75) family mediates suramin uptake while the AP-1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H+-ATPases to pentamidine action, and trypanothione and multiple putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of anti-trypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented levels of molecular insight into the mode of action of anti-trypanosomal drugs.

Graphical abstract Genome-scale RNA interference (RNAi) library screens in bloodstream-form Trypanosoma brucei, using nifurtimox and benznidazole, revealed type I nitroreductase, NTR, knockdown. A similar screen with eflornithine, revealed amino-acid transporter, AAT6, knockdown. . Research highlights ▶ A genome-scale RNA interference library is used to identify loss-of-function resistance mechanisms in bloodstream-form Trypanosoma brucei. ▶ Screens for resistance to nifurtimox or benznidazole identified loss of nitroreductase function. ▶ A screen for resistance to eflornithine identified loss of amino-acid transporter function., To be effective, therapeutic compounds must typically enter target cells and, in some cases, must be concentrated or modified. Thus, uptake and activation mechanisms often form the basis of selectivity against infectious agents. Loss-of-function screens can be used to identify proteins involved in drug uptake and metabolism and may also identify clinically relevant potential resistance mechanisms. We used a genome-scale RNA interference (RNAi) library to identify loss-of-function resistance mechanisms in bloodstream-form Trypanosoma brucei. Nifurtimox–Eflornithine Combination Therapy (NECT) was recently introduced for Human African Trypanosomiasis and we focus on these drugs here. Screens for resistance to nifurtimox and a related drug, benznidazole, identified loss of nitroreductase (NTR) pro-drug activator function. A screen for resistance to the amino-acid analogue, eflornithine, identified loss of amino-acid transporter (AAT6) function. Our results confirm recent findings and suggest that NTR or AAT6 loss-of-function represent major potential mechanisms of resistance to these drugs. Thus, bloodstream-form T. brucei RNAi libraries present a versatile tool for selective genetic screening and for the rapid identification of drug-activation, uptake and potential resistance mechanisms.

Background A diagnosis of cancer and anticipated death of a loved one has a significant impact on the whole family. Research has mainly focused on carers, with little emphasis on the wider, long-term implications. Aim To explore the cancer beliefs of patients with advanced cancer and their relatives. The focus was on their lived experiences and how these affected their beliefs, attitudes and constructions of cancer risk. Methods 27 in-depth, semi-structured interviews were conducted with advanced breast, colorectal or lung cancer patients and their close relatives. Interviews were recorded and transcribed verbatim. Data was analysed using the constant comparison method. Results A core category of fear, helplessness and fatalism emerged from the data. Family history was the most salient cancer risk factor and a diagnosis of advanced cancer increased perceptions of vulnerability for first-degree relatives. For relatives, the uncertainty and chaotic loss of control that accompanied an advanced cancer diagnosis resulted in multiple levels of fear and intensely negative or fatalistic attitudes to cancer. In contrast, patients held less negative views of cancer. They described several means of regaining control, including the importance of leaving a legacy – the hope that their situation would have a positive impact on others in the future. Conclusion Despite the prominence of ‘prevention’ in definitions of palliative care, services have evolved that are largely professional led and reactive. Adopting a health promoting approach based on empowerment is important, not just for improving care, but also increasing perceptions of control that may reduce negative cancer beliefs.

Making Sense of War provides a comprehensive and clear analysis of the complex business of waging war. It gives readers a thorough understanding of the key concepts in strategic thought, concepts that have endured since the Athenian general Thucydides and the Chinese philosopher/warrior Sun Tzu first wrote about strategy some 2500 years ago. It also examines the influence on strategic choice and military strategy of political, legal and technological change. This book discusses strategy at every level of competition, employing a thematic approach and using historical examples from 500 BCE to the present. It discusses the contraints and opportunities facing military commanders in the 21st century, and demonstrates that the formulation of military strategy will continue to be perhaps the single most important responsibility for senior security officials. Making Sense of War offers original insights into the imperatives of military success in the era of asymmetric warfare.

Acinetobacter species are ubiquitous Gram-negative bacteria widespread in nature. In the clinical setting, they have been isolated from the hospital environment and from the skin of health care workers. Although initially considered nonpathogenic, certain species of the genus Acinetobacter, particularly A. baumannii, are increasingly recognized as important nosocomial pathogens, particularly in intensive care unit (ICU) settings. With the increased use of invasive clinical procedures and expanding number of ICU beds, the prevalence of Acinetobacter infections is increasing. Also, there has been a rapid increase in resistance to all the major classes of antibiotics for Acinetobacter. Controlling the spread of Acinetobacter spp and the management of infections caused by these organisms poses a major challenge for the future.

Background It is widely accepted that a diagnosis of cancer affects the entire family. However, research has centred on primary caregivers, and little is known about the impact on the wider family. The literature has concentrated on female relatives of breast cancer patients. Studies involving relatives of colorectal cancer patients have focused on their attitudes to screening, or behavioural interventions, and have not attempted to identify their experiences, beliefs or needs. Aims To gain an in depth understanding of the experiences, beliefs and needs of first-degree relatives of patients diagnosed with advanced colorectal cancer. Methods Semi-structured interviews were conducted with adult children or siblings of patients with advanced colorectal cancer, referred to a palliative care service. Interviews explored their experience of a cancer diagnosis in the family and their beliefs about cancer. Data was transcribed verbatim and analysed qualitatively using a constant comparison method. Results 5 categories emerged from the data; ▸1) Emotional burden - The impact of witnessing a loved one with progressive disease was enormous. Relatives described the “shadow” cancer cast over the whole family, and feelings of fear on multiple levels. Emotional challenges were even greater when needing to support several generations within the family. ▸2) Practical burden, which was high even if they did not have a caring role, or live locally. ▸3) Changing family roles accompanying the anticipated death of their relative. ▸4) Feeling “out of the loop” and a need for more information. ▸5) Awareness of own risk – Some relatives described an increased sense of their personal risk of cancer, which exacerbated their fears and represented a significant barrier to open communication in the family. Conclusion This study highlights the profound impact of advanced colorectal cancer on the wider family, and suggests a role for palliative care in identifying and supporting their unmet needs.

There is increasing interest globally in the concept of ‘health promoting palliative care’ which aims to incorporate public health messages into palliative care services. However, there has been little emphasis on the prevention of ill health in palliative care. The role of palliative care in providing family-focused support, throughout a life limiting illness and into bereavement, means that opportunities to promote health in relatives and carers may be greater than in many other settings. Being a carer and subsequent bereavement can negatively impact on physical and emotional health. Furthermore, relatives of cancer patients are at increased risk of cancer themselves through shared genetic, environmental and lifestyle factors. While studies have evaluated family caregivers9 requirements in terms of psychological support and information, none have focused on addressing their physical and emotional health needs. Few studies have looked at information needs from the wider family perspective. We performed a literature review, pilot questionnaire study involving patients, carers and hospice staff (for which MREC permission was obtained) and family focus groups, to explore the potential of a palliative care, family-focused health promotion programme. The literature describes leaving a ‘healthy legacy’ as an important part of fostering hope in cancer and palliative care patients. Patients in our pilot study and focus groups described concerns for their family9s health and valued opportunities to discuss these. It was found that relatives and carers would welcome more information regarding their health and emotional wellbeing, but concerns were raised particularly by hospice staff. We will use the results of our investigations to inform further qualitative studies and to define the potential benefits of, and barriers to a health promotion programme aimed at families of palliative care patients.

EDITOR—Amoore and Ingram report that 400 people a year are seriously injured or killed as a result of adverse incidents with medical devices.1 We believe that this figure is the tip of the iceberg and that many more cases occur that are simply not recognised. Infectious complications of medical devices are often not considered in the context of reporting, and so the possible lessons that can minimise recurrence remain unlearnt. One of the most commonly used medical devices in hospital patients are peripheral intravenous …

Summary The neuraminidase, the membrane protein, the ribonucleoprotein (nucleocapsid protein) and the light and heavy polypeptide chains of the haemagglutinin subunits were isolated from particles of influenza virus type A0 (strain bel) and type B (strain lee). Each of these substances migrated as a single component during electrophoresis on SDS-polyacrylamide gels. The neuraminidase of type A2 influenza was isolated from the A0-A2 recombinant virus, X-7F1. This enzyme contained two polypeptides differing slightly in electrophoretic mobility. This paper gives the amino acid composition of the individual polypeptides from the type A0 and type B influenza viruses and the neuraminidase protein from X-7F1 virus.