Substitution of Met-38 to Ile in γ-synuclein found in two patients with amyotrophic lateral sclerosis induces aggregation into amyloid.

α-, ß-, and γ-Synuclein are intrinsically disordered proteins implicated in physiological processes in the nervous system of vertebrates. α-synuclein (αSyn) is the amyloidogenic protein associated with Parkinson's disease and certain other neurodegenerative disorders. Intensive research has focused on the mechanisms that cause αSyn to form amyloid structures, identifying its NAC region as being necessary and sufficient for amyloid assembly. Recent work has shown that a 7-residue sequence (P1) is necessary for αSyn amyloid formation. Although γ-synuclein (γSyn) is 55% identical in sequence to αSyn and its pathological deposits are also observed in association with neurodegenerative conditions, γSyn is resilient to amyloid formation in vitro. Here, we report a rare single nucleotide polymorphism (SNP) in the SNCG gene encoding γSyn, found in two patients with amyotrophic lateral sclerosis (ALS). The SNP results in the substitution of Met38 with Ile in the P1 region of the protein. These individuals also had a second, common and nonpathological, SNP in SNCG resulting in the substitution of Glu110 with Val. In vitro studies demonstrate that the Ile38 variant accelerates amyloid fibril assembly. Contrastingly, Val110 retards fibril assembly and mitigates the effect of Ile38. Substitution of residue 38 with Leu had little effect, while Val retards, and Ala increases the rate of amyloid formation. Ile38 γSyn also results in the formation of γSyn-containing inclusions in cells. The results show how a single point substitution can enhance amyloid formation of γSyn and highlight the P1 region in driving amyloid formation in another synuclein family member.

Medical leadership training varies substantially between UK medical schools: Report of the leadership in undergraduate medical education national survey (LUMENS).

BACKGROUND: Doctors are increasingly expected to demonstrate medical leadership and management (MLM) skills. The Faculty of Medical Leadership and Management (FMLM) has published an indicative undergraduate curriculum to guide the development of MLM content at UK medical schools. METHOD: Students from 30 medical schools were surveyed to determine their understanding of MLM teaching at their school. Timetables for 21 schools were searched for MLM-related keywords. Student-reported teaching and timetabled teaching were coded according to predefined themes. Aggregated demographic and postgraduate performance data were obtained through collaboration with the Medical Student Investigators Collaborative (msico.org). RESULTS: Whilst 88% of medical students see MLM teaching as relevant, only 18% believe it is well integrated into their curriculum. MLM content represented ∼2% of timetabled teaching in each 5-year undergraduate medical course. Most of this teaching was dedicated to teamwork, performance/reflection and communication skills. There was minimal association between how much of a topic students believed they were taught, and how much they were actually taught. We found no association between the volume of MLM teaching and performance in postgraduate examinations, trainee career destinations or fitness to practice referrals. CONCLUSION: Our findings demonstrate limited and variable teaching of MLM content. Delivery was independent of broader teaching and assessment factors.

An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory.

The common view is that T lymphocytes activate telomerase to delay senescence. Here we show that some T cells (primarily naïve and central memory cells) elongated telomeres by acquiring telomere vesicles from antigen-presenting cells (APCs) independently of telomerase action. Upon contact with these T cells, APCs degraded shelterin to donate telomeres, which were cleaved by the telomere trimming factor TZAP, and then transferred in extracellular vesicles at the immunological synapse. Telomere vesicles retained the Rad51 recombination factor that enabled telomere fusion with T-cell chromosome ends lengthening them by an average of ~3,000 base pairs. Thus, there are antigen-specific populations of T cells whose ageing fate decisions are based on telomere vesicle transfer upon initial contact with APCs. These telomere-acquiring T cells are protected from senescence before clonal division begins, conferring long-lasting immune protection.

Recruitment of inflammatory monocytes by senescent fibroblasts inhibits antigen-specific tissue immunity during human aging.

We have previously shown that healthy older adults exhibit reduced cutaneous immune responses during a varicella zoster virus (VZV) antigen challenge that correlated with a nonspecific inflammatory response to the injection itself. Here we found that needle damage during intradermal injections in older adults led to an increase in the number of cutaneous senescent fibroblasts expressing CCL2, resulting in the local recruitment of inflammatory monocytes. These infiltrating monocytes secreted prostaglandin E2, which inhibited resident memory T cell activation and proliferation. Pretreatment of older participants with a p38 mitogen-activated protein kinase inhibitor in vivo decreased CCL2 expression and inhibited monocyte recruitment and secretion of prostaglandin E2. This coincided with an increased response to VZV antigen challenge in the skin. Our results point to a series of molecular and cellular mechanisms that link cellular senescence, tissue damage, excessive inflammation and reduced immune responsiveness in human skin and demonstrate that tissue-specific immunity can be restored in older adults by short-term inhibition of inflammatory responses.

The Analysis of Teaching of Medical Schools (AToMS) survey: an analysis of 47,258 timetabled teaching events in 25 UK medical schools relating to timing, duration, teaching formats, teaching content, and problem-based learning.

BACKGROUND: What subjects UK medical schools teach, what ways they teach subjects, and how much they teach those subjects is unclear. Whether teaching differences matter is a separate, important question. This study provides a detailed picture of timetabled undergraduate teaching activity at 25 UK medical schools, particularly in relation to problem-based learning (PBL). METHOD: The Analysis of Teaching of Medical Schools (AToMS) survey used detailed timetables provided by 25 schools with standard 5-year courses. Timetabled teaching events were coded in terms of course year, duration, teaching format, and teaching content. Ten schools used PBL. Teaching times from timetables were validated against two other studies that had assessed GP teaching and lecture, seminar, and tutorial times. RESULTS: A total of 47,258 timetabled teaching events in the academic year 2014/2015 were analysed, including SSCs (student-selected components) and elective studies. A typical UK medical student receives 3960 timetabled hours of teaching during their 5-year course. There was a clear difference between the initial 2 years which mostly contained basic medical science content and the later 3 years which mostly consisted of clinical teaching, although some clinical teaching occurs in the first 2 years. Medical schools differed in duration, format, and content of teaching. Two main factors underlay most of the variation between schools, Traditional vs PBL teaching and Structured vs Unstructured teaching. A curriculum map comparing medical schools was constructed using those factors. PBL schools differed on a number of measures, having more PBL teaching time, fewer lectures, more GP teaching, less surgery, less formal teaching of basic science, and more sessions with unspecified content. DISCUSSION: UK medical schools differ in both format and content of teaching. PBL and non-PBL schools clearly differ, albeit with substantial variation within groups, and overlap in the middle. The important question of whether differences in teaching matter in terms of outcomes is analysed in a companion study (MedDifs) which examines how teaching differences relate to university infrastructure, entry requirements, student perceptions, and outcomes in Foundation Programme and postgraduate training.

Publisher Correction: Blocking elevated p38 MAPK restores efferocytosis and inflammatory resolution in the elderly.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Blocking elevated p38 MAPK restores efferocytosis and inflammatory resolution in the elderly.

Increasing age alters innate immune-mediated responses; however, the mechanisms underpinning these changes in humans are not fully understood. Using a human dermal model of acute inflammation, we found that, although inflammatory onset is similar between young and elderly individuals, the resolution phase was substantially impaired in elderly individuals. This arose from a reduction in T cell immunoglobulin mucin receptor-4 (TIM-4), a phosphatidylserine receptor expressed on macrophages that enables the engulfment of apoptotic bodies, so-called efferocytosis. Reduced TIM-4 in elderly individuals was caused by an elevation in macrophage p38 mitogen-activated protein kinase (MAPK) activity. Administering an orally active p38 inhibitor to elderly individuals rescued TIM-4 expression, cleared apoptotic bodies and restored a macrophage resolution phenotype. Thus, inhibiting p38 in elderly individuals rejuvenated their resolution response to be more similar to that of younger people. This is the first resolution defect identified in humans that has been successfully reversed, thereby highlighting the tractability of targeting pro-resolution biology to treat diseases driven by chronic inflammation.

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis.

Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56dim  CD57bright ) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+  CD57+ ) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.

Senescent cells evade immune clearance via HLA-E-mediated NK and CD8+ T cell inhibition.

Senescent cells accumulate in human tissues during ageing and contribute to age-related pathologies. The mechanisms responsible for their accumulation are unclear. Here we show that senescent dermal fibroblasts express the non-classical MHC molecule HLA-E, which interacts with the inhibitory receptor NKG2A expressed by NK and highly differentiated CD8+ T cells to inhibit immune responses against senescent cells. HLA-E expression is induced by senescence-associated secretary phenotype-related pro-inflammatory cytokines, and is regulated by p38 MAP kinase signalling in vitro. Consistently, HLA-E expression is increased on senescent cells in human skin sections from old individuals, when compared with those from young, and in human melanocytic nevi relative to normal skin. Lastly, blocking the interaction between HLA-E and NKG2A boosts immune responses against senescent cells in vitro. We thus propose that increased HLA-E expression contributes to persistence of senescent cells in tissues, thereby suggesting a new strategy for eliminating senescent cells during ageing.

CD56-negative NK cells with impaired effector function expand in CMV and EBV co-infected healthy donors with age.

Natural killer cells lacking expression of CD56 (CD56neg NK cells) have been described in chronic HIV and hepatitis C virus infection. Features and functions of CD56neg NK cells in the context of latent infection with CMV and / or EBV with age are not known. In a cohort of healthy donors >60 years of age, we found that co-infection with CMV and EBV drives expansion of CD56neg NK cells. Functionally, CD56neg NK cells displayed reduced cytotoxic capacity and IFN-γ production, a feature that was enhanced with CMV / EBV co-infection. Further, the frequency of CD56neg NK cells correlated with accumulation of end-stage-differentiated T cells and a reduced CD4 / CD8 T cell ratio, reflecting an immune risk profile. CD56neg NK cells had a mature phenotype characterized by low CD57 and KIR expression and lacked characteristics of cell senescence. No changes in their activating NK cell receptor expression, and no upregulation of the negative co-stimulation receptors PD-1 or TIM-3 were observed. In all, our data identify expansion of dysfunctional CD56neg NK cells in CMV+EBV+ elderly individuals suggesting that these cells may function as shape-shifters of cellular immunity and argue for a previously unrecognized role of EBV in mediating immune risk in the elderly.

Pneumothorax secondary to pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis (PAM) is a rare impairment of pulmonary phosphate clearance that leads to gradual precipitation of intra-alveolar calcium phosphate microliths. There is often a striking difference between alarming clinical imaging and a relatively well patient. Pneumothorax in PAM often only respond to surgical intervention.

Circulating Senescent T Cells Are Linked to Systemic Inflammation and Lesion Size During Human Cutaneous Leishmaniasis.

Leishmania (Viannia) braziliensis induces American tegumentary leishmaniasis that ranges in severity from the milder form, cutaneous (CL) to severe disseminated cutaneous leishmaniasis. Patients with CL develop a cell-mediated Th1 immune response accompanied by production of inflammatory cytokines, which contribute to parasite control and pathogenesis of disease. Here, we describe the accumulation of circulating T cells with multiple features of telomere dependent-senescence including elevated expression of CD57, KLRG-1, and γH2AX that have short telomeres and low hTERT expression during cutaneous L. braziliensis infection. This expanded population of T cells was found within the CD45RA+CD27- (EMRA) subset and produced high levels of inflammatory cytokines, analogous to the senescence-associated secretory profile (SASP) that has been described in senescent non-lymphoid cells. There was a significant correlation between the accumulation of these cells and the extent of systemic inflammation, suggesting that they are involved in the inflammatory response in this disease. Furthermore, these cells expressed high level of the skin homing receptor CLA and there was a highly significant correlation between the number of these cells in the circulation and the size of the Leishmania-induced lesions in the skin. Collectively our results suggest that extensive activation during the early stages of leishmaniasis drives the senescence of T cells with the propensity to home to the skin. The senescence-related inflammatory cytokine secretion by these cells may control the infection but also contribute to the immunopathology in the disease.

Colonic perforation by an intrathecal baclofen pump catheter causing delayed Escherichia coli meningitis.

Intrathecal baclofen (ITB) delivery via an implanted pump is frequently used for the treatment of spasticity. This is an effective and safe neurosurgical and pharmacological intervention associated with an improvement in patient quality of life. There is, however, a risk of device-related infection. We present a patient with pump-site infection and Escherichia coli meningitis secondary to transcolonic perforation of an intrathecal baclofen pump catheter. While this is rare, we review the intraoperative precautions and best practices that should be taken to prevent and manage this unusual complication.

Leadership and management in UK medical school curricula.

Purpose Although medical leadership and management (MLM) is increasingly being recognised as important to improving healthcare outcomes, little is understood about current training of medical students in MLM skills and behaviours in the UK. The paper aims to discuss these issues. Design/methodology/approach This qualitative study used validated structured interviews with expert faculty members from medical schools across the UK to ascertain MLM framework integration, teaching methods employed, evaluation methods and barriers to improvement. Findings Data were collected from 25 of the 33 UK medical schools (76 per cent response rate), with 23/25 reporting that MLM content is included in their curriculum. More medical schools assessed MLM competencies on admission than at any other time of the curriculum. Only 12 schools had evaluated MLM teaching at the time of data collection. The majority of medical schools reported barriers, including overfilled curricula and reluctance of staff to teach. Whilst 88 per cent of schools planned to increase MLM content over the next two years, there was a lack of consensus on proposed teaching content and methods. Research limitations/implications There is widespread inclusion of MLM in UK medical schools' curricula, despite the existence of barriers. This study identified substantial heterogeneity in MLM teaching and assessment methods which does not meet students' desired modes of delivery. Examples of national undergraduate MLM teaching exist worldwide, and lessons can be taken from these. Originality/value This is the first national evaluation of MLM in undergraduate medical school curricula in the UK, highlighting continuing challenges with executing MLM content despite numerous frameworks and international examples of successful execution.

Unusual placement of intrathecal baclofen pumps: report of two cases.

Intrathecal baclofen delivery via implantable pump represents an important modality for symptomatic relief in patients with chronic spasticity. Pumps are routinely implanted subcutaneously in the anterior abdominal wall. We describe two unusual cases where skin-related complications necessitated revision surgery in order to relocate the pump to alternative sites. The first patient was an international power canoeist, whose strenuous exercise programme interfered with his pump's original siting. The second patient was a cachectic university student with a history of cerebral palsy, who maintained low body mass despite attempted weight gain. The relocation of these two intrathecal devices to the medial compartment of the right thigh and right iliac fossa, respectively, is described.

Assessment at UK medical schools varies substantially in volume, type and intensity and correlates with postgraduate attainment.

BACKGROUND: In the United Kingdom (UK), medical schools are free to develop local systems and policies that govern student assessment and progression. Successful completion of an undergraduate medical degree results in the automatic award of a provisional licence to practice medicine by the General Medical Council (GMC). Such a licensing process relies heavily on the assumption that individual schools develop similarly rigorous assessment policies. Little work has evaluated variability of undergraduate medical assessment between medical schools. That absence is important in the light of the GMC's recent announcement of the introduction of the UKMLA (UK Medical Licensing Assessment) for all doctors who wish to practise in the UK. The present study aimed to quantify and compare the volume, type and intensity of summative assessment across medicine (A100) courses in the United Kingdom, and to assess whether intensity of assessment correlates with the postgraduate attainment of doctors from these schools. METHODS: Locally knowledgeable students in each school were approached to take part in guided-questionnaire interviews via telephone or Skype(TM). Their understanding of assessment at their medical school was probed, and later validated with the assessment department of the respective medical school. We gathered data for 25 of 27 A100 programmes in the UK and compared volume, type and intensity of assessment between schools. We then correlated these data with the mean first-attempt score of graduates sitting MRCGP and MRCP(UK), as well as with UKFPO selection measures. RESULTS: The median written assessment volume across all schools was 2000 min (mean = 2027, SD = 586, LQ = 1500, UQ = 2500, range = 1000-3200) and 1400 marks (mean = 1555, SD = 463, LQ = 1200, UQ = 1800, range = 1100-2800). The median practical assessment volume was 400 min (mean = 472, SD = 207, LQ = 400, UQ = 600, range = 200-1000). The median intensity (minutes per mark ratio) of summative written assessment was 1.24 min per mark (mean = 1.28, SD = 0.30, LQ = 1.11, UQ = 1.37, range = 0.85-2.08). An exploratory analysis suggested a significant correlation of total assessment time with mean first-attempt score on both the knowledge and the clinical assessments of MRCGP and of MRCP(UK). CONCLUSIONS: There are substantial differences in the volume, format and intensity of undergraduate assessment between UK medical schools. These findings suggest a potential for differences in the reliability of detecting poorly performing students, or differences in identifying and stratifying academically equivalent students for ranking in the Foundation Programme Application System (FPAS). Furthermore, these differences appear to directly correlate with performance in postgraduate examinations. Taken together, our findings highlight highly variable local assessment procedures that warrant further investigation to establish their potential impact on students.

Evaluation of bioluminescence-based assays of anti-malarial drug activity.

BACKGROUND: Transgenic Plasmodium falciparum expressing luciferase offers an attractive bioluminescence-based assay platform for the investigation of the pharmacological properties of anti-malarial drugs. Here a side-by-side comparison of bioluminescence and fluorescence-based assays, utilizing a luciferase reporter cassette that confers a strong temporal pattern of luciferase expression during the S-phase of intraerythrocytic development, is reported. METHODS: Key assay parameters for a range of commercially available luminogenic substrates are determined and compared to those measured using a Malaria Sybr Green I fluorescence assay. In addition, the short-term temporal effects of anti-malarial compounds are evaluated using both bioluminescent and fluorescent assay platforms. RESULTS: The Z', % coefficient of variation and 50% inhibition concentrations are essentially the same for bioluminescent and fluorescent assays in transgenic parasites generated in both chloroquine-sensitive and -resistant genetic backgrounds. Bioluminescent assays, irrespective of the luminogenic agent employed, do, however, offer significantly enhanced signal-to-noise ratios. Moreover, the bioluminescent assay is more dynamic in terms of determining temporal effects immediately following drug perturbation. CONCLUSION: This study suggests that opportunities for bioluminescence-based assays lie not in the measurement of 50% inhibition concentrations, where the cheaper fluorescence assay performs excellently and is not restricted by the need to genetically modify the parasite clone under investigation. Instead, assays that use the dynamic response of the luciferase reporter for semi-automated screening of additional pharmacological properties, such as relative rate-of-kill and lethal dose estimation, are a more attractive development opportunity.

Synthesis and evaluation of a novel series of indoloisoquinolines as small molecule anti-malarial leads.

A group of novel synthetic indoloisoquinolines was prepared and its potential as a novel series of small-molecule anti-malarial leads was assessed. The structure-activity relationship on variation of three distinct regions of chemical space was investigated. A lead compound was generated with an activity close to that observed for a known anti-malarial natural product, dihydrousambarensine, that shares the indoloisoquinoline template structure.