A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

The function of synaptotagmin as a Ca²⁺ sensor in neurotransmitter release involves Ca²⁺-dependent phospholipid binding to its two C₂ domains, but this activity alone does not explain why Ca²⁺ binding to the C₂B domain is more critical for release than Ca²⁺ binding to the C₂A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca²⁺ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca²⁺ binding to the C₂B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C₂B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.     

Development and Psychometric Evaluation of an Instrument to Assess Cross-Cultural Competence of Healthcare Professionals (CCCHP)

Background

Cultural competence of healthcare professionals (HCPs) is recognized as a strategy to reduce cultural disparities in healthcare. However, standardised, valid and reliable instruments to assess HCPs’ cultural competence are notably lacking. The present study aims to 1) identify the core components of cultural competence from a healthcare perspective, 2) to develop a self-report instrument to assess cultural competence of HCPs and 3) to evaluate the psychometric properties of the new instrument.

Methods

The conceptual model and initial item pool, which were applied to the cross-cultural competence instrument for the healthcare profession (CCCHP), were derived from an expert survey (n = 23), interviews with HCPs (n = 12), and a broad narrative review on assessment instruments and conceptual models of cultural competence. The item pool was reduced systematically, which resulted in a 59-item instrument. A sample of 336 psychologists, in advanced psychotherapeutic training, and 409 medical students participated, in order to evaluate the construct validity and reliability of the CCCHP.

Results

Construct validity was supported by principal component analysis, which led to a 32-item six-component solution with 50% of the total variance explained. The different dimensions of HCPs’ cultural competence are: Cross-Cultural Motivation/Curiosity, Cross-Cultural Attitudes, Cross-Cultural Skills, Cross-Cultural Knowledge/Awareness and Cross-Cultural Emotions/Empathy. For the total instrument, the internal consistency reliability was .87 and the dimension’s Cronbach’s α ranged from .54 to .84. The discriminating power of the CCCHP was indicated by statistically significant mean differences in CCCHP subscale scores between predefined groups.

Conclusions

The 32-item CCCHP exhibits acceptable psychometric properties, particularly content and construct validity to examine HCPs’ cultural competence. The CCCHP with its five dimensions offers a comprehensive assessment of HCPs’ cultural competence, and has the ability to distinguish between groups that are expected to differ in cultural competence. This instrument can foster professional development through systematic self-assessment and thus contributes to improve the quality of patient care.     

A potent peptidomimetic inhibitor of botulinum neurotoxin serotype A has a very different conformation than SNAP-25 substrate

Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (K(i)=41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the "proton shuttle" E224. This mechanism of inhibition is aided by residue contacts in the conserved S1' pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2' residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.     

Recombination provides evidence for ancient hybridisation in the <Emphasis Type="Italic">Silene aegyptiaca</Emphasis> (Caryophyllaceae) complex

Recombination events among distinct alleles complicate phylogenetic estimation. Various in vivo and in vitro processes can bring distinct alleles into the same genome to then undergo recombination, which may subsequently mislead phylogenetic inference if not assessed properly. Among the processes bringing divergent alleles together, hybridisation is perhaps the simplest and most likely, but alternatives need to be considered before hybridisation can be accepted as the underlying cause. Such alternatives include the presence of paralogues or deeply coalescing alleles, as well as amplification artefacts. Here, we document a recombination event that apparently took place between two divergent lineages of the Silene aegyptiaca complex in the flowering plant family Caryophyllaceae. We evaluate several possible mechanisms that might be responsible for the observed pattern. An ancient introgressive hybridisation event was the simplest explanation for the observations, compatible with geographic proximity of the affected lineages, whereas paralogy and deep coalescence are difficult to reconcile with the evidence obtained from a species tree of the group based on six different, non-recombinant genes and gene trees inferred using two partitions of the recombinant locus.     

Docking studies on monoamine oxidase-B inhibitors: estimation of inhibition constants (K(i)) of a series of experimentally tested compounds

Monoamine oxidase (EC1.4.3.4; MAO) is a mitochondrial outer membrane flavoenzyme that catalyzes the oxidation of biogenic amines. It has two distinct isozymic forms designated MAO-A and MAO-B, each displaying different substrate and inhibitor specificities. They are the well-known targets for antidepressant and neuroprotective drugs. Elucidation of the X-ray crystallographic structure of MAO-B has opened the way for molecular modeling studies. A series of experimentally tested (1-10) model compounds has been docked computationally to the active site of the MAO-B enzyme. The AutoDock 3.0.5 program was employed to perform automated molecular docking. The free energies of binding (DeltaG) and inhibition constants (K(i)) of the docked compounds were calculated by the Lamarckian Genetic Algorithm (LGA) of AutoDock 3.0.5. Excellent to good correlations between the calculated and experimental K(i) values were obtained.     

The N-terminal domains of syntaxin 7 and vti1b form three-helix bundles that differ in their ability to regulate SNARE complex assembly

The SNAREs syntaxin 7, syntaxin 8, vti1b, and endobrevin/VAMP8 function in the fusion of late endosomes. Although the core complex formed by these SNAREs is very similar to the neuronal SNARE complex, it differs from the neuronal complex in that three of the four SNAREs contain extended N-terminal regions of unknown structure and function. Here we show that the N-terminal regions of syntaxin 7, syntaxin 8, and vti1b contain well folded alpha-helical domains. Multidimensional NMR spectroscopy revealed that in syntaxin 7 and vti1b, the domains form three-helix bundles resembling those of syntaxin 1, Sso1p, and Vam3p. The three-helix bundle domain of vti1b is the first of its kind identified in a SNARE outside the syntaxin family. Only syntaxin 7 adopts a closed conformation, whereas in vti1b and syntaxin 8, the N-terminal domains do not interact with the adjacent SNARE motifs. Accordingly, the rate of SNARE complex assembly is retarded about 7-fold when syntaxin 7 contains its N-terminal domain, whereas the N-terminal domains of vti1b and syntaxin 8 have no influence on assembly kinetics. We conclude that three-helix bundles represent a common fold for SNARE N-terminal domains, not restricted to the syntaxin family. However, they differ in their ability to adopt closed conformations and thus to regulate the assembly of SNARE complexes.     

Endocytosis of synaptotagmin 1 is mediated by a novel,tryptophan-containing motif

The rate at which a membrane protein is internalized from the plasma membrane can be regulated by revealing a latent internalization signal in response to an appropriate stimulus. Internalization of the synaptic vesicle membrane protein, synaptotagmin 1, is controlled by two distinct regions of its intracytoplasmic C2B domain, an internalization signal present in the 29 carboxyterminal (CT) amino acids and a separate regulatory region. We have now characterized the internalization motif by mutagenesis and found that it involves an essential tryptophan in the last beta strand of the C2B domain, a region that is distinct from the AP2-binding site previously described. Internalization through the tryptophan-based motif is sensitive to eps15 and dynamin mutants and is therefore likely to be clathrin mediated. A tryptophan-to-phenylalanine mutation had no effect on internalization of the CT domain alone, but completely inhibited endocytosis of the folded C2B domain. This result suggests that recognition of sorting motifs can be influenced by their structural context. We conclude that endocytosis of synaptotagmin 1 requires a novel type of internalization signal that is subject to regulation by the rest of the C2B domain.     

Antimicrobial susceptibilities of Bacteroides fragilis and Bacteroides thetaiotaomicron strains isolated from clinical specimens and human intestinal microbiota

Species of Bacteroides fragilis group bacteria are the most prevalent pathogens and have the highest resistance rates to antimicrobial agents among anaerobic bacteria. Infections due to these micro-organisms often originate from patient's own intestinal microbiota. The objective of the study was to determine and compare the susceptibility profiles of clinical and intestinal B. fragilis and B. thetaiotaomicron strains against certain antimicrobials. Isolates were identified by conventional methods and API-20 A. Susceptibility tests were performed according to recommendations of NCCLS (M 11-A4) agar dilution methods. Beta-lactamase production was determined with nitrocefin discs. Forty-five clinical isolates (33 B. fragilis and 12 B. thetaiotaomicron) were from following sites: blood (n:8), intra-abdominal abscess (n:7), soft tissue (n:26), and miscellaneous foci of infection (n:4). Fifty B. fragilis and 60 B. thetaiotaomicron isolates from intestinal microbiota of individuals with no history of antimicrobial treatment within last 30 days were also examined. Beta-lactamase production was detected in 93% of clinical and 99% of intestinal isolates. The organisms including intestinal isolates were uniformly susceptible to metronidazole. The MIC90s of other antibiotics and resistance rates of all clinical isolates to those antibiotics were as follows: 256 microg/mL (93%) for ampicillin, 128 microg/mL (13%) for piperacillin, 64 microg/mL (11%) for cefoxitin, 1 microg/mL (2%) for amoxicillin-clavulanate, 0.5 microg/mL (2%) for imipenem, >256 microg/mL (36%) for clindamycin, 8 microg/mL (2%) for chloramphenicol. Intestinal isolates demonstrated similar resistance rates and MIC90s. Metronidazole, imipenem, amoxicillin-clavulanate seem to be effective drugs against these bacteria in Turkey.     

Streptosporangium anatoliense sp. nov., isolated from soil in Turkey

A novel actinobacterium, strain N9999^T, was isolated from soil and its taxonomic position determined using a polyphasic approach. The organism formed abundant aerial hyphae that differentiated into spherical spore vesicles. The cell wall contained meso-diaminopimelic acid; the whole-cell sugars were galactose, glucose, mannose, madurose and ribose; the predominant menaquinones MK-9 (H₂) and MK-9 (H₄); the major phospholipids phosphatidylethanolamine, diphosphatidylglycerol, a phosphaglycolipid and phosphatidylinositol mannosides; while the cellular fatty acids were rich in iso-C_14:0, C_15:0, cis-9-C_17:1, iso-C_16:0 and 10-methyl C_17:0 components. Phylogenetic analyses based on an almost complete 16S rRNA gene sequence indicated that strain N9999^T was closely related to a group that consisted of Streptosporangium pseudovulgare DSM 43181^T and Streptosporangium nondiastaticum DSM 43848^T. However, DNA–DNA relatedness and phenotypic data demonstrated that strain N9999^T was clearly distinguished from all closely related Streptosporangium species. The combined genotypic and phenotypic data demonstrate conclusively that the isolate should be classified as a new species of Streptosporangium.