Cellular and viral peptides bind multiple sites on the N-terminal domain of clathrin

Short peptide motifs in unstructured regions of clathrin-adaptor proteins recruit clathrin to membranes to facilitate post-Golgi membrane transport. Three consensus clathrin-binding peptide sequences have been identified and structural studies show that each binds distinct sites on the clathrin heavy chain N-terminal domain (NTD). A fourth binding site for adaptors on NTD has been functionally identified but not structurally characterised. We have solved high resolution structures of NTD bound to peptide motifs from the cellular clathrin adaptors β2 adaptin and amphiphysin plus a putative viral clathrin adaptor, hepatitis D virus large antigen (HDAg-L). Surprisingly, with each peptide we observe simultaneous peptide binding at multiple sites on NTD and viral peptides binding to the same sites as cellular peptides. Peptides containing clathrin-box motifs (CBMs) with the consensus sequence LΦxΦ[DE] bind at the 'arrestin box' on NTD, between β-propeller blades 4 and 5, which had previously been thought to bind a distinct consensus sequence. Further, we structurally define the fourth peptide binding site on NTD, which we term the Royle box. In vitro binding assays show that clathrin is more readily captured by cellular CBMs than by HDAg-L, and site-directed mutagenesis confirms that multiple binding sites on NTD contribute to efficient capture by CBM peptides.We thank Diamond Light Source for access to beamlines I02 and I04-1 (mx8547 and mx11235), this access being supported in part by the EU FP7 infrastructure grant BIOSTRUCT-X (contract no. 283570). This work was supported by a Sir Henry Dale Fellowship, jointly funded by the Royal Society and the Wellcome Trust, to S.C.G. (098406/Z/12/Z), by an NIH R01 grant (GM106963; L.M.T.) and by a Wellcome grant (090909/Z/09/Z; B.T.K.). J.M. holds a Wellcome Trust studentship. CIMR is supported by a Wellcome Trust Strategic Award (079895)

An automated multiwell plate reading film microscope for live cell autofluorescence lifetime assays

Fluorescence lifetime imaging (FLIM) is increasingly used to read out cellular autofluorescence originating from the coenzyme NADH in the context of investigating cell metabolic state. We present here an automated multiwell plate reading FLIM microscope optimized for UV illumination with the goal of extending high content fluorescence lifetime assays to readouts of metabolism. We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation. In particular, we illustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators, thereby illustrating the potential of the instrument for cytotoxicity studies, assays for drug discovery and stratified medicine

Development of mental health first aid guidelines on how a member of the public can support a person affected by a traumatic event: a Delphi study

BACKGROUND: People who experience traumatic events have an increased risk of developing a range of mental disorders. Appropriate early support from a member of the public, whether a friend, family member, co-worker or volunteer, may help to prevent the onset of a mental disorder or may minimise its severity. However, few people have the knowledge and skills required to assist. Simple guidelines may help members of the public to offer appropriate support when it is needed. METHODS: Guidelines were developed using the Delphi method to reach consensus in a panel of experts. Experts recruited to the panels included 37 professionals writing, planning or working clinically in the trauma area, and 17 consumer or carer advocates who had been affected by traumatic events. As input for the panels to consider, statements about how to assist someone who has experienced a traumatic event were sourced through a systematic search of both professional and lay literature. These statements were used to develop separate questionnaires about possible ways to assist adults and to assist children, and panel members answered either one questionnaire or both, depending on experience and expertise. The guidelines were written using the items most consistently endorsed by the panels across the three Delphi rounds. RESULTS: There were 180 items relating to helping adults, of which 65 were accepted, and 155 items relating to helping children, of which 71 were accepted. These statements were used to develop the two sets of guidelines appended to this paper. CONCLUSIONS: There are a number of actions which may be useful for members of the public when they encounter someone who has experienced a traumatic event, and it is possible that these actions may help prevent the development of some mental health problems in the future. Positive social support, a strong theme in these guidelines, has some evidence for effectiveness in developing mental health problems in people who have experienced traumatic events, but the degree to which it helps has not yet been adequately demonstrated. An evaluation of the effectiveness of these guidelines would be useful in determining their value. These guidelines may be useful to organisations who wish to develop or revise curricula of mental health first aid and trauma intervention training programs and policies. They may also be useful for members of the public who want immediate information about how to assist someone who has experienced a potentially traumatic event

Estimating the prevalence and predictors of musculoskeletal disorders in Tanzania: a cross-sectional pilot study

\ua9 2024, African Field Epidemiology Network. All rights reserved.Introduction: musculoskeletal (MSK) disorders account for approximately 20% of all years lived with disability worldwide however studies of MSK disorders in Africa are scarce. This pilot study aimed to estimate the community-based prevalence of MSK disorders, identify predictors, and assess the associated disability in a Tanzanian population. Methods: a cross-sectional study was conducted in one village in the Kilimanjaro region from March to June 2019. The Gait, Arms, Legs, Spine (GALS) or paediatric GALS (pGALS) examinations were used during household and school visits. Individuals positive in GALS/pGALS screening were assessed by the regional examination of the musculoskeletal system (REMS) and Modified Health Assessment Questionnaire (MHAQ). Results: among the 1,172 individuals enrolled in households, 95 (8.1%, 95% CI: 6.6-9.8) showed signs of MSK disorders using the GALS/pGALS examination and 37 (3.2%, 95% CI: 2.2-4.3) using the REMS. Among 682 schools enrolled children, seven showed signs of MSK disorders using the GALS/pGALS examination (1.0%, 95% CI: 0.4-2.1) and three using the REMS (0.4%, 95% CI: 0.0-1.3). In the household-enrolled adult population, female gender and increasing age were associated with GALS and REMS-positive findings. Among GALS-positive adults, increasing age was associated with REMS-positive status and increasing MHAQ score. Conclusion: this Tanzanian study demonstrates a prevalence of MSK disorders and identifies predictors of MSK disorders comparable to those seen globally. These findings can inform the development of rheumatology services and interventions in Tanzania and the design of future investigations of the determinants of MSK disorders, and their impacts on health, livelihoods, and well-being

Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation.

CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role

Monitoring postmatch fatigue during a competitive season in elite youth soccer players

Context: Countermovement jump (CMJ) and perceived wellness measures are useful for monitoring fatigue. Fatigue indicators should simultaneously show sensitivity to previous load and demonstrate influence on subsequent physical output; however, these factors have not been examined. Objective: To explore the efficacy of CMJ and wellness measures to both detect postmatch fatigue and predict subsequent physical match output in elite youth soccer players. Design: Cross-sectional study. Patients or Other Participants: Sixteen soccer players (18 ± 1 years) from 36 English Football League Youth Alliance League fixtures. Main Outcome Measure(s): Physical match outputs (total distance, high-speed running, very high-speed running, and accelerations and decelerations [AD]) were recorded using a 10-Hz global positioning system and 200-Hz accelerometer device during competitive match play. The CMJ height and perceived wellness were assessed weekly and daily, respectively, as indirect indicators of fatigue. Four subunits of wellness (perceived soreness, energy, general stress, and sleep) were measured using customized psychometric questionnaires. Results: Simple linear regression showed that match AD predicted energy (R 2 = 0.08, P =.001), stress (R 2 = 0.09, P<.001), and total wellness (R 2 = 0.06, P =.002) at 2 days postmatch. The CMJ (R 2 = 0.05, P =.002), stress (R 2 = 0.08, P<.001), sleep (R 2 = 0.03, P =.034), and total wellness (R 2 = 0.05, P =.006) measures at 5 days prematch predicted AD during the subsequent match. Conclusions: The CMJ and wellness measures may be useful for detecting postmatch fatigue. Wellness scores, but not CMJ, at 5 days prematch influenced subsequent match output and therefore may be used to plan and periodize training for the upcoming microcycle

Fine sediment reduces vertical migrations of Gammarus pulex (Crustacea: Amphipoda) in response to surface water loss

Surface and subsurface sediments in river ecosystems are recognized as refuges that may promote invertebrate survival during disturbances such as floods and streambed drying. Refuge use is spatiotemporally variable, with environmental factors including substrate composition, in particular the proportion of fine sediment (FS), affecting the ability of organisms to move through interstitial spaces. We conducted a laboratory experiment to examine the effects of FS on the movement of Gammarus pulex Linnaeus (Crustacea: Amphipoda) into subsurface sediments in response to surface water loss. We hypothesized that increasing volumes of FS would impede and ultimately prevent individuals from migrating into the sediments. To test this hypothesis, the proportion of FS (1–2 mm diameter) present within an open gravel matrix (4–16 mm diameter) was varied from 10 to 20% by volume in 2.5% increments. Under control conditions (0% FS), 93% of individuals moved into subsurface sediments as the water level was reduced. The proportion of individuals moving into the subsurface decreased to 74% at 10% FS, and at 20% FS no individuals entered the sediments, supporting our hypothesis. These results demonstrate the importance of reducing FS inputs into river ecosystems and restoring FS-clogged riverbeds, to promote refuge use during increasingly common instream disturbances

The incidence of total hip arthroplasty after hip arthroscopy in osteoarthritic patients

<p>Abstract</p> <p>Objective</p> <p>To assess the incidence of total hip arthroplasty (THA) in osteoarthritic patients who were treated by arthroscopic debridement and to evaluate factors that might influence the time interval from the first hip arthroscopy to THA.</p> <p>Design</p> <p>Retrospective clinical series</p> <p>Methods</p> <p>Follow-up data and surgical reports were retrieved from 564 records of osteoarthritic patients that have had hip arthroscopy between the years 2002 to 2009 with a mean follow-up time of 3.2 years (range, 1-6.4 years). The time interval between the first hip arthroscopy to THA was modelled as a function of patient age; level of cartilage damage; procedures performed and repeated arthroscopies with the use of multivariate regression analysis.</p> <p>Results</p> <p>Ninety (16%) of all participants eventually required THA. The awaiting time from the first arthroscopy to a hip replacement was found to be longer in patients younger than 55 years and in a milder osteoarthritic stage. Patients that experienced repeated hip scopes had a longer time to THA than those with only a single procedure. Procedures performed concomitant with debridement and lavage did not affect the time interval to THA.</p> <p>Conclusions</p> <p>In our series of arthroscopic treatment of hip osteoarthritis, 16% required THA over a period of 7 years. Factors that influence the time to arthroplasty were age, degree of osteoarthritis and recurrent procedures.</p

Pneumonic Tularemia in Rabbits Resembles the Human Disease as Illustrated by Radiographic and Hematological Changes after Infection

Background: Pneumonic tularemia is caused by inhalation of the gram negative bacterium, Francisella tularensis. Because of concerns that tularemia could be used as a bioterrorism agent, vaccines and therapeutics are urgently needed. Animal models of pneumonic tularemia with a pathophysiology similar to the human disease are needed to evaluate the efficacy of these potential medical countermeasures. Principal Findings: Rabbits exposed to aerosols containing Francisella tularensis strain SCHU S4 developed a rapidly progressive fatal pneumonic disease. Clinical signs became evident on the third day after exposure with development of a fever (>40.5°C) and a sharp decline in both food and water intake. Blood samples collected on day 4 found lymphopenia and a decrease in platelet counts coupled with elevations in erythrocyte sedimentation rate, alanine aminotransferase, cholesterol, granulocytes and monocytes. Radiographs demonstrated the development of pneumonia and abnormalities of intestinal gas consistent with ileus. On average, rabbits were moribund 5.1 days after exposure; no rabbits survived exposure at any dose (190-54,000 cfu). Gross evaluation of tissues taken at necropsy showed evidence of pathology in the lungs, spleen, liver, kidney and intestines. Bacterial counts confirmed bacterial dissemination from the lungs to the liver and spleen. Conclusions/Significance: The pathophysiology of pneumonic tularemia in rabbits resembles what has been reported for humans. Rabbits therefore are a relevant model of the human disease caused by type A strains of F. tularensis. © 2011 Reed et al