Intraflagellar transport speed is sensitive to genetic and mechanical perturbations to flagellar beating.

Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule doublets to periodically bend the flagellum for beating, while intraflagellar transport (IFT) kinesins and dyneins carry IFT trains bidirectionally along the axoneme. Despite assembling motile cilia and flagella, IFT train speeds have only previously been quantified in immobilized flagella-mechanical immobilization or genetic paralysis. This has limited investigation of the interaction between IFT and flagellar beating. Here, in uniflagellate Leishmania parasites, we use high-frequency, dual-color fluorescence microscopy to visualize IFT train movement in beating flagella. We discovered that adhesion of flagella to a microscope slide is detrimental, reducing IFT train speed and increasing train stalling. In flagella free to move, IFT train speed is not strongly dependent on flagella beat type; however, permanent disruption of flagella beating by deletion of genes necessary for formation or regulation of beating showed an inverse correlation of beat frequency and IFT train speed.

Activation of Nod2 signaling upon norovirus infection enhances antiviral immunity and susceptibility to colitis.

Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.

Radial spoke protein 9 is necessary for axoneme assembly in Plasmodium but not in trypanosomatid parasites.

Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The '9+2' axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella.

Starr-Edwards mitral valve-48 years till redo surgery, with over 54 years of patient survival after primary mitral valve replacement.

We report a male patient who underwent successful redo cardiac surgery in 2014; tricuspid valve repair and redo mitral valve replacement of a Starr-Edwards mitral valve implanted 48 years previously. Six years after the redo operation, the patient remains well and therefore has an impressive over 54 years of survival following mitral valve replacement surgery.

Multifaceted modes of action of the anticancer probiotic Enterococcus hirae.

A deviated repertoire of the gut microbiome predicts resistance to cancer immunotherapy. Enterococcus hirae compensated cancer-associated dysbiosis in various tumor models. However, the mechanisms by which E. hirae restored the efficacy of cyclophosphamide administered with concomitant antibiotics remain ill defined. Here, we analyzed the multifaceted modes of action of this anticancer probiotic. Firstly, E. hirae elicited emigration of thymocytes and triggered systemic and intratumoral IFNγ-producing and CD137-expressing effector memory T cell responses. Secondly, E. hirae activated the autophagy machinery in enterocytes and mediated ATG4B-dependent anticancer effects, likely as a consequence of its ability to increase local delivery of polyamines. Thirdly, E. hirae shifted the host microbiome toward a Bifidobacteria-enriched ecosystem. In contrast to the live bacterium, its pasteurized cells or membrane vesicles were devoid of anticancer properties. These pleiotropic functions allow the design of optimal immunotherapies combining E. hirae with CD137 agonistic antibodies, spermidine, or Bifidobacterium animalis. We surmise that immunological, metabolic, epithelial, and microbial modes of action of the live E. hirae cooperate to circumvent primary resistance to therapy.

Cross-reactivity between tumor MHC class I-restricted antigens and an enterococcal bacteriophage.

Intestinal microbiota have been proposed to induce commensal-specific memory T cells that cross-react with tumor-associated antigens. We identified major histocompatibility complex (MHC) class I-binding epitopes in the tail length tape measure protein (TMP) of a prophage found in the genome of the bacteriophage Enterococcus hirae Mice bearing E. hirae harboring this prophage mounted a TMP-specific H-2Kb-restricted CD8+ T lymphocyte response upon immunotherapy with cyclophosphamide or anti-PD-1 antibodies. Administration of bacterial strains engineered to express the TMP epitope improved immunotherapy in mice. In renal and lung cancer patients, the presence of the enterococcal prophage in stools and expression of a TMP-cross-reactive antigen by tumors correlated with long-term benefit of PD-1 blockade therapy. In melanoma patients, T cell clones recognizing naturally processed cancer antigens that are cross-reactive with microbial peptides were detected.

Evaluation of completeness of resection of the glandular portion of the uterus with hysterectomy in dogs - A pilot study.

Gonadectomies are the most commonly performed surgical procedures in dogs. The benefits and risks associated with gonadectomy in dogs have been broadly investigated. Due to concerns associated with ovariectomy, some pet-owners and veterinarians are considering surgical alternatives which terminate the capacity for reproduction without removal of the gonads to preserve functions of the gonadal hormones. The objective of this study was to determine the feasibility of complete resection of the glandular portion of the uterus with hysterectomy (HE). Using a prospective cadaveric and an ex-vivo design, uterine resection was performed cranially and caudally to the cervix, and at the proper ligament to simulate routine ovariohysterectomy and HE. Using microscopic evaluation procedures, there was identification of glandular tissue at the surgical margin of all samples resected cranially to the cervix and no glandular tissue in the samples resected caudally to the cervix. Half of the samples from the uterine horns contained glandular tissue. The results of the present study indicate ligation and transection of the uterus caudally to the cervix (as recommended for HE) allows for complete resection of glandular tissues, however, transection at the proper ligament may not. The clinical relevance of retention of para-ovarian uterine glandular tissue is unknown. Additional research is needed where there is investigation of the consequences and surgical technique for HE before there is widespread clinical use of this procedure.

Therapeutics-how to treat phase separation-associated diseases.

Liquid-liquid phase separation has drawn attention as many neurodegeneration or cancer-associated proteins are able to form liquid membraneless compartments (condensates) by liquid-liquid phase separation. Furthermore, there is rapidly growing evidence that disease-associated mutation or post-translational modification of these proteins causes aberrant location, composition or physical properties of the condensates. It is ambiguous whether aberrant condensates are always causative in disease mechanisms, however they are likely promising potential targets for therapeutics. The conceptual framework of liquid-liquid phase separation provides opportunities for novel therapeutic approaches. This review summarises how the extensive recent advances in understanding control of nucleation, growth and composition of condensates by protein post-translational modification has revealed many possibilities for intervention by conventional small molecule enzyme inhibitors. This includes the first proof-of-concept examples. However, understanding membraneless organelle formation as a physical chemistry process also highlights possible physicochemical mechanisms of intervention. There is huge demand for innovation in drug development, especially for challenging diseases of old age including neurodegeneration and cancer. The conceptual framework of liquid-liquid phase separation provides a new paradigm for thinking about modulating protein function and is very different from enzyme lock-and-key or structured binding site concepts and presents new opportunities for innovation.

Lysosome assembly and disassembly changes endocytosis rate through the Leishmania cell cycle.

The Leishmania lysosome has an atypical structure, consisting of an elongated vesicle-filled tubule running along the anterior-posterior axis of the cell, which is termed the multivesicular tubule (MVT) lysosome. Alongside, the MVT lysosome is one or more microtubules, the lysosomal microtubule(s). Previous work indicated there were cell cycle-related changes in MVT lysosome organization; however, these only provided snapshots and did not connect the changes in the lysosomal microtubule(s) or lysosomal function. Using mNeonGreen tagged cysteine peptidase A and SPEF1 as markers of the MVT lysosome and lysosomal microtubule(s), we examined the dynamics of these structures through the cell cycle. Both the MVT lysosome and lysosomal microtubule(s) elongated at the beginning of the cell cycle before plateauing and then disassembling in late G2 before cytokinesis. Moreover, the endocytic rate in cells where the MVT lysosome and lysosomal microtubule(s) had disassembled was extremely low. The dynamic nature of the MVT lysosome and lysosomal microtubule(s) parallels that of the Trypanosoma cruzi cytostome/cytopharynx, which also has a similar membrane tubule structure with associated microtubules. As the cytostome/cytopharynx is an ancestral feature of the kinetoplastids, this suggests that the Leishmania MVT lysosome and lysosomal microtubule(s) are a reduced cytostome/cytopharynx-like feature.

Bioprosthetic mitral valve thrombosis and subsequent obstruction from non-Hodgkins lymphoma despite anticoagulation.

Malignancy can cause a hypercoagulable state, it rarely causes prosthetic valve thrombosis, with only limited cases reports due to lymphoma. Although no standardised approach for treatment exists, the combination of early diagnosis, surgical intervention and adjuvant chemotherapy are likely to provide the best chances of survival. We describe an interesting case of a bioprosthetic mitral valve thrombosis leading to valve obstruction in a patient with non-Hodgkin's lymphoma despite being on anticoagulation.

Direction of flagellum beat propagation is controlled by proximal/distal outer dynein arm asymmetry.

The 9 + 2 axoneme structure of the motile flagellum/cilium is an iconic, apparently symmetrical cellular structure. Recently, asymmetries along the length of motile flagella have been identified in a number of organisms, typically in the inner and outer dynein arms. Flagellum-beat waveforms are adapted for different functions. They may start either near the flagellar tip or near its base and may be symmetrical or asymmetrical. We hypothesized that proximal/distal asymmetry in the molecular composition of the axoneme may control the site of waveform initiation and the direction of waveform propagation. The unicellular eukaryotic pathogens Trypanosoma brucei and Leishmania mexicana often switch between tip-to-base and base-to-tip waveforms, making them ideal for analysis of this phenomenon. We show here that the proximal and distal portions of the flagellum contain distinct outer dynein arm docking-complex heterodimers. This proximal/distal asymmetry is produced and maintained through growth by a concentration gradient of the proximal docking complex, generated by intraflagellar transport. Furthermore, this asymmetry is involved in regulating whether a tip-to-base or base-to-tip beat occurs, which is linked to a calcium-dependent switch. Our data show that the mechanism for generating proximal/distal flagellar asymmetry can control waveform initiation and propagation direction.

Myocardial ischaemia as a result of external coronary compression from infective aortic root aneurysm: atypical presentation of prosthetic valve endocarditis.

SummaryThis case describes an unusual presentation of prosthetic valve endocarditis (PVE): an acute coronary syndrome. A 67-year-old male presented with cardiac sounding chest pain on a background of a short history of night sweats, weight loss and general malaise. Four months previously, he had undergone bio-prosthetic aortic valve replacement for severe aortic stenosis and single vessel bypass grafting of the obtuse marginal. Whilst having chest pain, his ECG showed infero-lateral ST depression. Early coronary angiography revealed a new right coronary artery (RCA) lesion that was not present prior to his cardiac surgery. Using multi-modality cardiac imaging, the diagnosis of PVE was made. An aortic root abscess was demonstrated that was causing external compression of the RCA. LEARNING POINTS: PVE accounts for up to 20% of all cases of infective endocarditis.High clinical suspicion and early blood cultures before empirical antibiotics are key as the presentation of PVE can often be atypical.PVE rarely presents as an acute coronary syndrome. Potential mechanisms by which PVE may result in an ACS include coronary embolization, obstruction of coronary ostia by a large mobile vegetation and external coronary artery compression from an infective aneurysms/abscess.Repeat cardiac surgery is often required for high-risk PVE such as those caused by staphylococcal infection or severe prosthetic dysfunction.

Diagnosis and assessment of dilated cardiomyopathy: a guideline protocol from the British Society of Echocardiography.

Heart failure (HF) is a debilitating and life-threatening condition, with 5-year survival rate lower than breast or prostate cancer. It is the leading cause of hospital admission in over 65s, and these admissions are projected to rise by more than 50% over the next 25 years. Transthoracic echocardiography (TTE) is the first-line step in diagnosis in acute and chronic HF and provides immediate information on chamber volumes, ventricular systolic and diastolic function, wall thickness, valve function and the presence of pericardial effusion, while contributing to information on aetiology. Dilated cardiomyopathy (DCM) is the third most common cause of HF and is the most common cardiomyopathy. It is defined by the presence of left ventricular dilatation and left ventricular systolic dysfunction in the absence of abnormal loading conditions (hypertension and valve disease) or coronary artery disease sufficient to cause global systolic impairment. This document provides a practical approach to diagnosis and assessment of dilated cardiomyopathy that is aimed at the practising sonographer.

Blocking Synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei Leads to an Increase in Macrophage Phagocytosis Due to Reduced Clearance of Surface Coat Antibodies.

The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a chronic infection.

A safety checklist for transoesophageal echocardiography from the British Society of Echocardiography and the Association of Cardiothoracic Anaesthetists.

The World Health Organisation (WHO) launched the Surgical Safety Checklist in 2008. The introduction of this checklist resulted in a significant reduction in the incidence of complications and death in patients undergoing surgery. Consequently, the WHO Surgical Safety checklist is recommended for use by the National Patient Safety Agency for all patients undergoing surgery. However, many invasive or interventional procedures occur outside the theatre setting and there are increasing requirements for a safety checklist to be used prior to such procedures. Transoesophageal echocardiography (TOE) is an invasive procedure and although generally considered to be safe, it carries the risk of serious and potentially life-threatening complications. Strict adherence to a safety checklist may reduce the rate of significant complications during TOE. However, the standard WHO Surgical Safety Checklist is not designed for procedures outside the theatre environment and therefore this document is designed to be a procedure-specific safety checklist for TOE. It has been endorsed for use by the British Society of Echocardiography and the Association of Cardiothoracic Anaesthetists.

Mycotic thoracic aortic arch aneurysm from haematogenous spread of Clostridium septicum due to metastatic colorectal cancer: a survival guide.

We report the case of a 78-year-old female who was found to have a mycotic thoracic aortic arch aneurysm caused by Clostridium septicum. Subsequent investigations demonstrated adenocarcinoma of the caecum with two liver metastases. The patient underwent a hybrid procedure involving endovascular repair of the aneurysm with a right-to-left carotid crossover bypass and a left carotid to left subclavian bypass to protect the cerebral and left subclavian blood flow. The caecal tumour was later completely excised by laparoscopic right hemicolectomy. The patient then underwent resection for the liver metastases. There was no evidence of tumour recurrence 2 years after her right hemicolectomy. This case emphasizes the importance of the association of C. septicum infection with colorectal malignancy. To our knowledge, this is the first case of a mycotic aortic arch aneurysm caused by C. septicum being successfully treated with a hybrid endovascular repair procedure.

Episodic circulatory collapse caused by intermittent prosthetic aortic valve dysfunction.

An 80-year-old woman with a 20-mm Medtronic Hall (tilting-disc) aortic prosthesis presented with episodes of chest pain associated with circulatory collapse and subsequent rapid spontaneous recovery. A computed tomography angiogram demonstrated no evidence of aortic dissection, pulmonary embolus, or coronary artery disease. A transthoracic echocardiogram demonstrated a well-functioning prosthetic aortic valve. After another inpatient episode, auscultation demonstrated an intermittent absence of the prosthetic second heart sound. A transesophageal echocardiography examination revealed that the aortic valve was intermittently sticking in the open position. During the emergency replacement, the existing valve was noted to have pannus and overlying thrombus, which are rare in this type of valve but can lead to dramatic circulatory collapse when present. This case demonstrates how a diagnosis of intermittent valve dysfunction can prove elusive. It is important, as part of the assessment of mechanical valves, to perform regular, careful auscultation and echocardiographic examination, with emphasis on recording in Doppler or M-mode through the valve at slow sweep speeds for a prolonged period in order to maximize the chance of diagnosing episodic valve dysfunction.

The trypanolytic factor-mechanism, impacts and applications.

The Trypanosoma brucei subspecies T. brucei brucei is non-human infective due to susceptibility to lysis by trypanolytic factor (TLF) in human serum. Reviewed here are the advances which have revealed apolipoprotein L1 (ApoL1), found in high density lipoprotein, as the lysis-inducing component of TLF, the means of uptake via haptoglobin-related protein receptor and the mechanism of resistance in T. b. rhodesiense via its serum resistance-associated (SRA) protein. The first practical steps to application of these discoveries are now in progress; transgenic animals expressing either baboon or minimally truncated human ApoL1 show resistance to both T. b. brucei and T. b. rhodesiense. This has major implications for treatment and prevention of human and animal African trypanosomiasis.

Human papillomavirus and survival of patients with oropharyngeal cancer.

BACKGROUND: Oropharyngeal squamous-cell carcinomas caused by human papillomavirus (HPV) are associated with favorable survival, but the independent prognostic significance of tumor HPV status remains unknown. METHODS: We performed a retrospective analysis of the association between tumor HPV status and survival among patients with stage III or IV oropharyngeal squamous-cell carcinoma who were enrolled in a randomized trial comparing accelerated-fractionation radiotherapy (with acceleration by means of concomitant boost radiotherapy) with standard-fractionation radiotherapy, each combined with cisplatin therapy, in patients with squamous-cell carcinoma of the head and neck. Proportional-hazards models were used to compare the risk of death among patients with HPV-positive cancer and those with HPV-negative cancer. RESULTS: The median follow-up period was 4.8 years. The 3-year rate of overall survival was similar in the group receiving accelerated-fractionation radiotherapy and the group receiving standard-fractionation radiotherapy (70.3% vs. 64.3%; P=0.18; hazard ratio for death with accelerated-fractionation radiotherapy, 0.90; 95% confidence interval [CI], 0.72 to 1.13), as were the rates of high-grade acute and late toxic events. A total of 63.8% of patients with oropharyngeal cancer (206 of 323) had HPV-positive tumors; these patients had better 3-year rates of overall survival (82.4%, vs. 57.1% among patients with HPV-negative tumors; P<0.001 by the log-rank test) and, after adjustment for age, race, tumor and nodal stage, tobacco exposure, and treatment assignment, had a 58% reduction in the risk of death (hazard ratio, 0.42; 95% CI, 0.27 to 0.66). The risk of death significantly increased with each additional pack-year of tobacco smoking. Using recursive-partitioning analysis, we classified our patients as having a low, intermediate, or high risk of death on the basis of four factors: HPV status, pack-years of tobacco smoking, tumor stage, and nodal stage. CONCLUSIONS: Tumor HPV status is a strong and independent prognostic factor for survival among patients with oropharyngeal cancer. (ClinicalTrials.gov number, NCT00047008.)