FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P2-dependent switch.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.

Near-Sun observations of an F-corona decrease and K-corona fine structure.

Remote observations of the solar photospheric light scattered by electrons (the K-corona) and dust (the F-corona or zodiacal light) have been made from the ground during eclipses1 and from space at distances as small as 0.3 astronomical units2-5 to the Sun. Previous observations6-8 of dust scattering have not confirmed the existence of the theoretically predicted dust-free zone near the Sun9-11. The transient nature of the corona has been well characterized for large events, but questions still remain (for example, about the initiation of the corona12 and the production of solar energetic particles13) and for small events even its structure is uncertain14. Here we report imaging of the solar corona15 during the first two perihelion passes (0.16-0.25 astronomical units) of the Parker Solar Probe spacecraft13, each lasting ten days. The view from these distances is qualitatively similar to the historical views from ground and space, but there are some notable differences. At short elongations, we observe a decrease in the intensity of the F-coronal intensity, which is suggestive of the long-sought dust free zone9-11. We also resolve the fine-scale plasma structure of very small eruptions, which are frequently ejected from the Sun. These take two forms: the frequently observed magnetic flux ropes12,16 and the predicted, but not yet observed, magnetic islands17,18 arising from the tearing-mode instability in the current sheet. Our observations of the coronal streamer evolution confirm the large-scale topology of the solar corona, but also reveal that, as recently predicted19, streamers are composed of yet smaller substreamers channelling continual density fluctuations at all visible scales.

A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation.

Besides AP-2 and clathrin triskelia, clathrin coat inception depends on a group of early-arriving proteins including Fcho1/2 and Eps15/R. Using genome-edited cells, we described the role of the unstructured Fcho linker in stable AP-2 membrane deposition. Here, expanding this strategy in combination with a new set of llama nanobodies against EPS15 shows an FCHO1/2-EPS15/R partnership plays a decisive role in coat initiation. A nanobody containing an Asn-Pro-Phe peptide within the complementarity-determining region 3 loop is a function-blocking pseudoligand for tandem EPS15/R EH domains. Yet, in living cells, EH domains gathered at clathrin-coated structures are poorly accessible, indicating residence by endogenous NPF-bearing partners. Forcibly sequestering cytosolic EPS15 in genome-edited cells with nanobodies tethered to early endosomes or mitochondria changes the subcellular location and availability of EPS15. This combined approach has strong effects on clathrin coat structure and function by dictating the stability of AP-2 assemblies at the plasma membrane.

Correction to: The help for people with money, employment or housing problems (HOPE) intervention: pilot randomised trial with mixed methods feasibility research.

[This corrects the article DOI: 10.1186/s40814-018-0365-6.].

The help for people with money, employment or housing problems (HOPE) intervention: pilot randomised trial with mixed methods feasibility research.

BACKGROUND: Job loss, austerity measures, financial difficulties and house repossession contribute to the risk of self-harm and suicide during recessions. Navigating the benefits system and accessing sources of welfare and debt advice is a difficult experience for vulnerable people, further contributing to their distress. Whilst there is some evidence that advice-type interventions can lead to financial gain, there is mixed evidence for their effectiveness in improving mental health in those experiencing financial difficulties. There have been no interventions targeting those who have self-harmed due to economic hardship. METHODS: Our aim was to determine the feasibility and acceptability of a brief psychosocial intervention (the 'HOPE' service) for people presenting to hospital emergency departments (ED) following self-harm or in acute distress because of financial, employment or welfare (benefit) difficulties. Nineteen people consented to random allocation to the intervention or control arm on a 2:1 basis. Participants randomised to the intervention arm (n = 13) received up to six sessions of 1:1 support provided by community support staff trained in Motivational Interviewing (MI). Control participants (n = 6) received a one-off session signposting them to relevant support organisations. Fourteen participants were followed up after 3 months. Participants and mental health workers took part in qualitative interviews. The acceptability of outcome measures including the PHQ-9, GAD-7, repeat self-harm, EQ5D-5 L and questions about debt, employment and welfare benefits were explored. RESULTS: Interviews indicated the main benefits of the service as the resolution of specific financial problems and receiving support when participants were feeling most vulnerable. Randomisation was acceptable to most participants although not always fully understood and control participants could be disappointed. Recruitment was slow (1-2 per month). The outcome measures were acceptable and appeared sensitive to change. DISCUSSION: The HOPE intervention is feasible and acceptable. There was evidence of need and it is a relatively inexpensive intervention. Refining aspects of the intervention would be straightforward. A full-scale RCT would be feasible, if broadened eligibility criteria led to increased recruitment and improvements were made to staff training and support. TRIAL REGISTRATION: ISRCTN58531248.

Angiotensin receptor blocker vs ACE inhibitor effects on HDL functionality in patients on maintenance hemodialysis.

BACKGROUND AND AIMS: Angiotensin receptor blockers (ARB) and angiotensin converting enzyme inhibitors (ACEI) reduce cardiovascular events in the general population. Maintenance hemodialysis (MHD) patients are at high cardiovascular risk but few studies have directly addressed the comparative efficacy of these drugs. MHD disrupts the normally atheroprotective actions of high density lipoprotein (HDL), therefore, we compared ACEI or ARB treatment on HDL functions in MHD. METHODS AND RESULTS: HDL was isolated at the starting point (pre) and 3-6 months later (post) in 30 MHD randomly assigned to placebo, ramipril or valsartan. Outcomes included cholesterol efflux, inflammatory cytokine response, effects on Toll-like receptors (TLR), superoxide production, methylarginine and serum amyloid A (SAA) levels. HDL from ARB- or ACEI-treated subjects was more effective in maintaining efflux than HDL of placebo. HDL from ARB- or ACEI-treated subjects but not placebo lessened cellular superoxide production. In contrast, neither ARB nor ACEI improved HDL anti-inflammatory effect. Indeed, HDL of ACEI-treated subjects potentiated the cytokine responses in association with activation of TLR but did not alter the HDL content of methylarginines or SAA. CONCLUSION: Both ACEI and ARB stabilized HDL cholesterol acceptor function and sustained cellular anti-oxidative effects but not anti-inflammatory effects, and ACEI-treatment instead amplified the HDL inflammatory response. The findings reveal possible utility of antagonizing angiotensin actions in MDH and suggest a possible mechanism for superiority of ARB vs ACEI in the setting of advanced kidney disease.

The Role of Twist in Kinked Flux Rope Emergence and Delta-spot Formation.

It has been observationally well established that the magnetic configurations most favorable for producing energetic flaring events reside in δ-spots, a class of sunspots defined as having opposite-polarity umbrae sharing a common penumbra. They are frequently characterized by extreme compactness, strong rotation, and anti-Hale orientation. Numerous studies have shown that nearly all of the largest solar flares originate in δ-spots, making the understanding of these structures a fundamental step in predicting space weather. Despite their important influence on the space environment, surprisingly little is understood about the origin and behavior of δ-spots. In this paper, we perform a systematic study of the behavior of emerging flux ropes to test a theoretical model for the formation of δ-spots: the kink instability of emerging flux ropes. We simulated the emergence of highly twisted, kink-unstable flux ropes from the convection zone into the corona, and we compared their photospheric properties to those of emerged weakly twisted, kink-stable flux ropes. We show that the photospheric manifestations of the emergence of highly twisted flux ropes closely match the observed properties of δ-spots, and we discuss the resulting implications for observations. Our results strongly support and extend previous theoretical work that suggested that the kink instability of emerging flux ropes is a promising candidate to explain δ-spot formation, as it reproduces their key characteristics very well.

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 ß2 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 ß-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.

A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenic Escherichia coli in beef and bovine faeces.

AIM: To develop a multiplex loop-mediated isothermal amplification (LAMP) assay capable of quantifying Escherichia coli and differentiating verocytotoxigenic E. coli (VTEC). METHODS AND RESULTS: Primer sets were selected to amplify the phoA gene (all E. coli strains) and stx1 and/or stx2 genes (VTEC strains only). LAMP calibration curves demonstrated good quantification capability compared with conventional culture. The limits of detection 50% (LOD50 ) of the multiplex LAMP assay were 2·8 (95% CI 2·4-3·3), 3·2 (95% CI 2·5-3·9) and 2·8-3·2 (95% CI 2·1-3·5) log CFU per g for the phoA, stx1 and stx2 genes, respectively. When validated by testing retail beef and bovine faeces samples, good correlation between E. coli counts indicated by the LAMP assay and culture was observed; however, false-negative LAMP assay results were obtained for 12·5-14·7% of samples. CONCLUSIONS: A rapid, multiplex LAMP assay for direct quantification of E. coli and specific detection of VTEC in beef and faeces was successfully developed. Further optimisation of the assay would be needed to improve detection sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex LAMP assay represents a rapid alternative to culture for monitoring E. coli levels on beef for hygiene monitoring purposes, and, potentially, a method for detection of VTEC in beef and faeces.

Transient Fcho1/2⋠Eps15/R⋠AP-2 Nanoclusters Prime the AP-2 Clathrin Adaptor for Cargo Binding.

Clathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 µ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement.

F-BAR/EFC Domain Proteins: Some Assembly Required.

Polymeric spirals of crescent-shaped BAR-domain superfamily proteins are touted to girdle eukaryotic phospholipid bilayers into narrow tubules for trafficking and membrane remodeling events. But McDonald et al. (2015) in this issue of Developmental Cell question whether this broadly held view and conceptually appealing mechanism for membrane sculpting is really overhyped.

Canine neural angiostrongylosis: a case-control study in Sydney dogs.

The editors wish to correct an error in the Results section. On page 197, third paragraph, lines 3­5 should read: Younger dogs were significantly (P<0.001) more likely to be cases than older dogs (odds ratio 11.8; 95% confidence interval 3.9­35.9). The editors sincerely apologise for this error.

Canine neural angiostrongylosis: a case-control study in Sydney dogs.

OBJECTIVE: To determine the risk factors for canine neural angiostrongylosis in dogs domiciled in Sydney, Australia; geographic location, age, sex, neuter status, weight and breed were assessed. PROCEDURE: Case and matched-control dogs were selected from three veterinary clinics in Sydney. Conditional logistic regression was used to identify factors associated with disease status. A scan statistic was used to identify disease clusters. RESULTS: Age (young dogs) and neuter status (entire dogs) were independent risk factors for neural angiostrongylosis diagnosis, and diagnoses predominantly occurred during autumn, with some evidence of spatial clustering. CONCLUSIONS: Veterinarians in endemic areas should be aware of these risk factors when presented with suspect canine neural angiostrongylosis cases and also should consider advising clients of preventive treatment. Potential human health risks should be further investigated, because urban dog populations might represent a useful sentinel species for disease in humans.

A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing.

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.

Visualization of clathrin-mediated endocytosis in live Drosophila egg chambers.

In oviparous animals, clathrin-dependent endocytosis is often critical to stockpile a necessary supply of yolk within the maturing oocyte, which enables subsequent embryonic development. In the physically linked chains of maturing egg chambers within the Drosophila melanogaster ovary, a distinct, morphologically discernable subset undergoes a massive burst clathrin-mediated endocytosis to accumulate yolk in a process termed vitellogenesis. Here, we describe how to prepare isolated ovaries to follow endocytosis, and detail approaches to follow live uptake of soluble reporters into vitellogenic Drosophila egg chambers.

A phosphotyrosine switch for cargo sequestration at clathrin-coated buds.

The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargo is directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargo for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 ß2 subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short α-helical interaction motif. This signal, found in the CLASPs ß-arrestin and the autosomal recessive hypercholesterolemia (ARH) protein, docks into an elongated groove on the ß2 appendage platform. Tyr-888 is a critical constituent of this spatially confined ß2 appendage contact interface and is phosphorylated in numerous high-throughput proteomic studies. We find that a phosphomimetic Y888E substitution does not interfere with incorporation of expressed ß2-YFP subunit into AP-2 or alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the ß2 appendage, indicating that the mutated appendage is folded and operational. However, the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and ß-arrestin. Phyogenetic conservation of Tyr-888 suggests that this residue can reversibly control occupancy of the ß2 platform-binding site and, hence, cargo sorting.

Cargo recognition in clathrin-mediated endocytosis.

The endosomal system is expansive and complex, characterized by swift morphological transitions, dynamic remodeling of membrane constituents, and intracellular positioning changes. To properly navigate this ever-altering membrane labyrinth, transmembrane protein cargoes typically require specific sorting signals that are decoded by components of protein coats. The best-characterized sorting process within the endosomal system is the rapid internalization of select transmembrane proteins within clathrin-coated vesicles. Endocytic signals consist of linear motifs, conformational determinants, or covalent modifications in the cytosolic domains of transmembrane cargo. These signals are interpreted by a diverse set of clathrin-associated sorting proteins (CLASPs) that translocate from the cytosol to the inner face of the plasma membrane. Signal recognition by CLASPs is highly cooperative, involving additional interactions with phospholipids, Arf GTPases, other CLASPs, and clathrin, and is regulated by large conformational changes and covalent modifications. Related sorting events occur at other endosomal sorting stations.

Prevalence of Epstein-Barr Virus in a population of patients with inflammatory bowel disease: a prospective cohort study.

BACKGROUND: The Epstein-Barr Virus (EBV) is truly prolific, with a prevalence of more than 90% in the adult human population. There are, however, little data available on the prevalence of EBV among patients with Inflammatory Bowel Disease (IBD), a population that is frequently immunosuppressed and thus at risk for severe, often fatal, primary infection. AIM: To identify the prevalence of EBV in a population of patients with IBD and to compare it with that of the general population. METHODS: A database of 2500 IBD patients previously followed at the University of Alberta IBD Centre was queried; 60 of these patients were randomly chosen to participate. A total of 220 patients attending the IBD Centre for clinical appointment were also prospectively asked to participate. Participants completed serological testing for VCA-IgM, VCA-IgG and EBNA-IgG, to determine prior EBV exposure. RESULTS: A total of 263 patients underwent testing. Results for EBV seroprevalence of specific age groups were as follows: 18-20 years (n = 17), 29% seronegative; 21-25 years (n = 31), 29% seronegative; 26-30 years (n = 35), 31-35 years (n = 18) and 36-40 years (n = 25), 100% seropositive. Finally, 3% of those older than 40 (n = 117) were seronegative. EBV seroprevalence was similar for Crohn's disease and ulcerative colitis. Azathioprine was associated with seropositivity (P = 0.048). CONCLUSION: The prevalence of EBV seronegativity in the IBD population aged 18-25 years was similar to that described in the general population, and above age 25 years, seropositivity approached 100%.