Ferlins Show Tissue-Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans-Golgi/Recycling Ferlins

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Ferlins are an ancient family of Ca2+-binding, multi-C2 domain vesicle fusion proteins. Of the six human ferlins, mutations in dysferlin cause muscular dystrophy and otoferlin cause deafness. We detail the tissue-distribution, subcellular localization and endocytic trafficking of the human ferlins. Dysferlin and myoferlin, type-I ferlins, localize to the plasma membrane and late endosomes, which display potential for occasional recycling. Otoferlin and Fer1L6, type-II ferlins, localize to dedicated recycling subcompartments of the trans-Golgi network. We establish that type-I and type-II ferlins segregate into late-endosomal and recycling trans-Golgi compartments. Ferlins are a family of transmembrane-anchored vesicle fusion proteins uniquely characterized by 5-7 tandem cytoplasmic C2 domains, Ca2+-regulated phospholipid-binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca2+-dependent, vesicle-mediated membrane repair and otoferlin mutations cause non-syndromic deafness due to defective Ca2+-triggered auditory neurotransmission. In this study, we describe the tissue-specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D-structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7-positive late endosomes, supporting potential roles in the late-endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans-Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans-Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type-I and type-II ferlins segregate as PM/late-endosomal or trans-Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations

Structurally Diverse Covalent Triazine-based Framework Materials for Photocatalytic Hydrogen Evolution from Water

A structurally diverse family of 39 covalent triazine-based framework materials (CTFs) are synthesized by Suzuki–Miyaura polycondensation and tested as hydrogen evolution photocatalysts using a high-throughput workflow. The two best-performing CTFs are based on benzonitrile and dibenzo[b,d]thiophene sulfone linkers, respectively, with catalytic activities that are among the highest for this material class. The activities of the different CTFs are rationalized in terms of four variables: the predicted electron affinity, the predicted ionization potential, the optical gap, and the dispersibility of the CTFs particles in solution, as measured by optical transmittance. The electron affinity and dispersibility in solution are found to be the best predictors of photocatalytic hydrogen evolution activity

Calpains, Cleaved Mini-Dysferlin(C72), and L-Type Channels Underpin Calcium-Dependent Muscle Membrane Repair

Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlin(C72). Mini-dysferlin(C72)-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlin(C72) and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein-or phospholipid-binding role for muscle membrane repair

Increasing the frequency of hand washing by healthcare workers does not lead to commensurate reductions in staphylococcal infection in a hospital ward

Hand hygiene is generally considered to be the most important measure that can be applied to prevent the spread of healthcare-associated infection (HAI). Continuous emphasis on this intervention has lead to the widespread opinion that HAI rates can be greatly reduced by increased hand hygiene compliance alone. However, this assumes that the effectiveness of hand hygiene is not constrained by other factors and that improved compliance in excess of a given level, in itself, will result in a commensurate reduction in the incidence of HAI. However, several researchers have found the law of diminishing returns to apply to hand hygiene, with the greatest benefits occurring in the first 20% or so of compliance, and others have demonstrated that poor cohorting of nursing staff profoundly influences the effectiveness of hand hygiene measures. Collectively, these findings raise intriguing questions about the extent to which increasing compliance alone can further reduce rates of HAI. In order to investigate these issues further, we constructed a deterministic Ross-Macdonald model and applied it to a hypothetical general medical ward. In this model the transmission of staphylococcal infection was assumed to occur after contact with the transiently colonized hands of HCWs, who, in turn, acquire contamination only by touching colonized patients. The aim of the study was to evaluate the impact of imperfect hand cleansing on the transmission of staphylococcal infection and to identify, whether there is a limit, above which further hand hygiene compliance is unlikely to be of benefit. The model demonstrated that if transmission is solely via the hands of HCWs, it should, under most circumstances, be possible to prevent outbreaks of staphylococcal infection from occurring at a hand cleansing frequencies <50%, even with imperfect hand hygiene. The analysis also indicated that the relationship between hand cleansing efficacy and frequency is not linear - as efficacy decreases, so the hand cleansing frequency required to ensure R0<1 increases disproportionately. Although our study confirmed hand hygiene to be an effective control measure, it demonstrated that the law of diminishing returns applies, with the greatest benefit derived from the first 20% or so of compliance. Indeed, our analysis suggests that there is little benefit to be accrued from very high levels of hand cleansing and that in most situations compliance >40% should be enough to prevent outbreaks of staphylococcal infection occurring, if transmission is solely via the hands of HCWs. Furthermore we identified a non-linear relationship between hand cleansing efficacy and frequency, suggesting that it is important to maximise the efficacy of the hand cleansing process

Weather-it missions: a social network analysis perspective of an online citizen inquiry community

Citizen inquiry is an innovative informal science learning approach, which engages members of the general public in scientific investigations sparked by their personal experience of everyday science, and to which other members can contribute. This paper aims to describe the network of interactions and contributions of Weather-it, an online Citizen Inquiry community accommodated by the nQuire-it platform, which involves people in creating and maintaining their own weather missions (investigations). The interaction patterns within Weather-it are mainly explored through social network analysis of community members and missions. The results indicate the quiet and active members within the community, their splitting into sub-communities, and their contribution and data collection methods and preferences. These results provide in-sight into the behaviour of people in such public engagement projects

Structure-activity relationships for analogs of the tuberculosis drug bedaquiline with the naphthalene unit replaced by bicyclic heterocycles

Replacing the naphthalene C-unit of the anti-tuberculosis drug bedaquiline with a range of bicyclic heterocycles of widely differing lipophilicity gave analogs with a 4.5-fold range in clogP values. The biological results for these compounds indicate on average a lower clogP limit of about 5.0 in this series for retention of potent inhibitory activity (MIC90s) against M.tb in culture. Some of the compounds also showed a significant reduction in inhibition of hERG channel potassium current compared with bedaquiline, but there was no common structural feature that distinguished these

The treatment of segmental tibial fractures: does patient preference differ from surgeon choice?

INTRODUCTION: Segmental tibial fractures are complex injuries with a prolonged recovery time. Current definitive treatment options include intramedullary fixation or a circular external fixator. However, there is uncertainty as to which surgical option is preferable and there are no sufficiently rigorous multi-centre trials that have answered this question. The objective of this study was to determine whether patient and surgeon opinion was permissive for a randomised controlled trial (RCT) comparing intramedullary nailing to the application of a circular external fixator. MATERIALS AND METHODS: A convenience questionnaire survey of attending surgeons was conducted during the United Kingdom's Orthopaedic Trauma Society annual meeting 2017 to determine the treatment modalities used for a segmental tibial fracture (n=63). Patient opinion was obtained from clinical patients who had been treated for a segmental tibial fracture as part of a patient and public involvement focus group with questions covering the domains of surgical preference, treatment expectations, outcome, the consent process and follow-up regime (n=5). RESULTS: Based on the surgeon survey, 39% routinely use circular frame fixation following segmental tibial fracture compared to 61% who use nail fixation. Nail fixation was reported as the treatment of choice for a closed injury in a healthy patient in 81% of surgeons, and by 86% for a patient with a closed fracture who was obese. Twenty-one percent reported that they would use a nail for an open segmental tibia fracture in diabetics who smoked, whilst 57% would opt for a nail for a closed injury with compartment syndrome, and only 27% would use a nail for an open segmental injury in a young fit sports person. The patient and public preference exercise identified that sleep, early functional outcomes and psychosocial measures of outcomes are important. CONCLUSION: We concluded that a RCT comparing definitive fixation with an intramedullary nail and a circular external fixator is justified as there remains uncertainty on the optimal surgical management for segmental tibial fractures. Furthermore, psychosocial factors and early post-operative outcomes should be reported as core outcome measures as part of such a trial

Solution structure of a repeated unit of the ABA-1 nematode polyprotein allergen of ascaris reveals a novel fold and two discrete lipid-binding sites

Parasitic nematode worms cause serious health problems in humans and other animals. They can induce allergic-type immune responses, which can be harmful but may at the same time protect against the infections. Allergens are proteins that trigger allergic reactions and these parasites produce a type that is confined to nematodes, the nematode polyprotein allergens (NPAs). These are synthesized as large precursor proteins comprising repeating units of similar amino acid sequence that are subsequently cleaved into multiple copies of the allergen protein. NPAs bind small lipids such as fatty acids and retinol (Vitamin A) and probably transport these sensitive and insoluble compounds between the tissues of the worms. Nematodes cannot synthesize these lipids, so NPAs may also be crucial for extracting nutrients from their hosts. They may also be involved in altering immune responses by controlling the lipids by which the immune and inflammatory cells communicate. We describe the molecular structure of one unit of an NPA, the well-known ABA-1 allergen of Ascaris and find its structure to be of a type not previously found for lipid-binding proteins, and we describe the unusual sites where lipids bind within this structur

Mammalian Comparative Sequence Analysis of the Agrp Locus

Agouti-related protein encodes a neuropeptide that stimulates food intake. Agrp expression in the brain is restricted to neurons in the arcuate nucleus of the hypothalamus and is elevated by states of negative energy balance. The molecular mechanisms underlying Agrp regulation, however, remain poorly defined. Using a combination of transgenic and comparative sequence analysis, we have previously identified a 760 bp conserved region upstream of Agrp which contains STAT binding elements that participate in Agrp transcriptional regulation. In this study, we attempt to improve the specificity for detecting conserved elements in this region by comparing genomic sequences from 10 mammalian species. Our analysis reveals a symmetrical organization of conserved sequences upstream of Agrp, which cluster into two inverted repeat elements. Conserved sequences within these elements suggest a role for homeodomain proteins in the regulation of Agrp and provide additional targets for functional evaluation

Finding and Resolving Security Misusability with Misusability Cases

Although widely used for both security and usability concerns, scenarios used in security design may not necessarily inform the design of usability, and vice- versa. One way of using scenarios to bridge security and usability involves explicitly describing how design deci- sions can lead to users inadvertently exploiting vulnera- bilities to carry out their production tasks. This paper describes how misusability cases, scenarios that describe how design decisions may lead to usability problems sub- sequently leading to system misuse, address this problem. We describe the related work upon which misusability cases are based before presenting the approach, and illus- trating its application using a case study example. Finally, we describe some findings from this approach that further inform the design of usable and secure systems