α-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate

α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson''s disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P₂). Our results show that α-Syn colocalizes with PIP₂ and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin-mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI(4,5)P₂ levels on the plasma membrane. In accord with their effects on PI(4,5)P₂ levels, the PD associated A30P, E46K, and A53T mutations in α-Syn further enhance CME in neuronal and nonneuronal cells. However, lysine to glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, which interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI(4,5)P₂ levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn–mediated membrane trafficking.     

Human immune response against salivary antigens of Simulium damnosum s.l.: A new epidemiological marker for exposure to blackfly bites in onchocerciasis endemic areas

BackgroundSimulium damnosum sensu lato (s.l.) blackflies transmit Onchocerca volvulus, a filarial nematode that causes human onchocerciasis. Human landing catches (HLCs) is currently the sole method used to estimate blackfly biting rates but is labour-intensive and questionable on ethical grounds. A potential alternative is to measure host antibodies to vector saliva deposited during bloodfeeding. In this study, immunoassays to quantify human antibody responses to S. damnosum s.l. saliva were developed, and the salivary proteome of S. damnosum s.l. was investigated.Methodology/Principal findingsBlood samples from people living in onchocerciasis-endemic areas in Ghana were collected during the wet season; samples from people living in Accra, a blackfly-free area, were considered negative controls and compared to samples from blackfly-free locations in Sudan. Blackflies were collected by HLCs and dissected to extract their salivary glands. An ELISA measuring anti-S. damnosum s.l. salivary IgG and IgM was optimized and used to quantify the humoral immune response of 958 individuals. Both immunoassays differentiated negative controls from endemic participants. Salivary proteins were separated by gel-electrophoresis, and antigenic proteins visualized by immunoblot. Liquid chromatography mass spectrometry (LC–MS/MS) was performed to characterize the proteome of S. damnosum s.l. salivary glands. Several antigenic proteins were recognized, with the major ones located around 15 and 40 kDa. LC–MS/MS identified the presence of antigen 5-related protein, apyrase/nucleotidase, and hyaluronidase.Conclusions/SignificanceThis study validated for the first time human immunoassays that quantify humoral immune responses as potential markers of exposure to blackfly bites. These assays have the potential to facilitate understanding patterns of exposure as well as evaluating the impact of vector control on biting rates. Future studies need to investigate seasonal fluctuations of these antibody responses, potential cross-reactions with other bloodsucking arthropods, and thoroughly identify the most immunogenic proteins.     

Phenotypic and genotypic characteristics and epidemiological significance of ctx+ strains of Vibrio cholerae isolated from seafood in Malaysia

Of 97 strains of Vibrio cholerae isolated from various seafoods in Malaysia in 1998 and 1999, 20 strains carried the ctx gene and produced cholera toxin. Fourteen, one, and five of these toxigenic strains belonged to the O139, O1 Ogawa, and rough serotypes, respectively. The rough strains had the rfb gene of the O1 serotype. The toxigenic strains varied in their biochemical characteristics, the amount of cholera toxin produced, their antibiograms, and the presence or absence of the pTLC plasmid sequence. DNA fingerprinting analysis by arbitrarily primed PCR, ribotyping, and a pulsed-field gel electrophoresis method classified the toxigenic strains into 3, 7, and 10 types, respectively. The relatedness of these toxigenic strains to clinical strains isolated in other countries and from international travelers was examined by using a dendrogram constructed from the pulsed-field gel electrophoresis profiles. The results of the examination of the antibiogram and the possession of the toxin-linked cryptic plasmid were consistent with the dendrogram-based relatedness: the O139 strains isolated from Malaysian seafoods could be separated into two groups that appear to have been introduced from the Bengal area independently. The rough strains of Malaysian seafood origin formed one group and belonged to a cluster unique to the Thailand-Malaysia-Laos region, and this group may have persisted in this area for a long period. The single O1 Ogawa strain detected in Malaysian seafood appears to have an origin and route of introduction different from those of the O139 and the rough strains.     

Use of randomly amplified polymorphic DNA analysis to differentiate isolates of Vibrio parahaemolyticus from cockles (Anadara granosa)

A total of 35 Kanagawa-negative strains of Vibrio parahaemolyticus isolated from cockles (Anadara granosa) were investigated by randomly amplified polymorphic DNA fingerprinting with three primers and their plasmid profiles. Eighteen strains carried small plasmid(s) of 2.4 to 7.3kb that enabled the V. parahaemolyticus to be grouped into eight plasmid patterns. The three primers generated polymorphisms in all 35 strains of V. parahaemolyticus tested, producing bands ranging from 0.25 to 3.9kb. The RAPD profiles revealed a high level of DNA sequence diversity within the Vibrio parahaemolyticus strains tested, and that cockles in the study area are populated by genetically polymorphic strains of V. parahaemolyticus.     

Effect of cup abduction angle and head lateral microseparation on contact stresses in ceramic-on-ceramic total hip arthroplasty

A finite elements model was developed in order to evaluate the combined influence of the head lateral microseparation and the cup abduction angle on the contact pressure in Ceramic-on-Ceramic Total Hip Arthroplasty. The model's parameters were those used on the Leeds II hip simulator. A 32 mm head diameter and a 30 μm radial clearance was used. The cup was positioned with an abduction angle ranging from 45° to 90°. The medio-lateral microseparation varied from 0 to 500 μm. A load of 2500 N was applied through the head centre. For 45° abduction angle, edge loading appeared above a medial-lateral separation of 30 μm. Complete edge loading was obtained for a 60 μm medial-lateral separation. Under edge loading conditions, the contact area was found to be elliptical. For 45° abduction angle, as the head lateral separation increased, the maximal contact pressure increased from 66 MPa and converged to an asymptotic value of 205 MPa. Both cup abduction and lateral microseparation displacement induced a large increase in the stresses in Ceramic-on-Ceramic THA. However, this increase in contact pressure induced by higher abduction angle, became negligible as the lateral separation increased.     

Sprouted and Freeze‐Dried Wheat and Oat Seeds – Phytochemical Profile and in Vitro Biological Activities

This research was carried out to study phytochemical profile, in vitro antioxidant capacity, reducing power, anti‐hyperglycemic, anti‐inflammatory activities and simulated gastrointestinal digestion of 7‐day old cereal sprouts: spelt wheat ‘Nirvana’ (WSSpe), wheat ‘Simonida’ (WSSim), oat ‘Golozrni’ (OSG) and oat ‘Jadar’ (OSJ). OSG expressed significantly higher (P ≤ 0.05) total phenols (TPC) and flavonoids content (TFC), antioxidant capacities (DPPH and ABTS assays) and reducing power (EC₅₀^DPPH = 2.12 mg/ml; EC₅₀^ABTS = 0.87 mg/ml; EC_0.5^RP = 12.24 mg/ml) as well as anti‐hyperglycemic activity (EC₅₀^AHgA = 0.96 mg/ml). WSSpe had the highest content of chlorophyll (131.23 mg/100 g) and carotenoids (22.84 mg/100 g). WSSim possessed the most potent anti‐inflammatory activity (2.71 mg/ml), though not significantly different from OSG (2.77 mg/ml). The in vitro simulation of gastro‐intestinal digestion showed higher release of phenolic compounds in intestinal than in gastric fluid.