Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites.     

GABARAPL1 antibodies: target one protein, get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.     

EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.     

Methicillin-susceptible ST398 Staphylococcus aureus responsible for bloodstream infections: an emerging human-adapted subclone?

In the course of an annual 3-month bloodstream infections (BSI) survey conducted during a four-year period in 31 healthcare institutions located in three noncontiguous French regions, we report 18 ST398 Staphylococcus aureus BSI. ST398 BSI incidence showed a seven-fold increase during the study period (0.002 per 1,000 patient days in 2007 vs. 0.014 in 2010). ST398 BSI isolates differed from the pig-borne multiresistant clone: 17/18 BSI isolates were methicillin susceptible and none was of t011, t034 or t108 pig-borne spa-types. ST398 BSI isolates had homogenous resistance patterns (15/18 with only Ery(r)) and prophagic content (all harboured the hlb-converting Sau3int phage). The clustering of BSI and pig-borne isolates by spa-typing and MLVA, the occurrence of Sau3int phage in BSI isolates and the lack of this phage in pig-borne isolates suggest that the emergence of BSI isolates could have arisen from horizontal transfer, at least of the Sau3int phage, in genetically diverse MSSA ST398 isolates. The acquisition of the phage likely plays a role in the increasing ability of the lysogenic ST398 isolates to colonize human. The mode of acquisition of the non pig-borne ST398 isolates by our 18 patients remains unclear. ST398 BSI were diagnosed in patients lacking livestock exposure and were significantly associated with digestive portals of entry (3/18 [16.7%] for ST398 vs. 19/767 [2.5%] for non ST398 BSI; p?=?.012). This raises the question of possible foodborne human infections. We suggest the need for active surveillance to study and control the spread of this human-adapted subclone increasingly isolated in the hospital setting.     

A state–space model to derive bluefin tuna movement and habitat from archival tags

In the echiuran worm Bonellia viridis Rolando, the vast majority of sexually undifferentiated larvae metamorphose into dwarf males that live inside the female when exposed to females, but differentiate into females when developing in the absence of females. By means of a spatially explicit, individual-based model we examine how this specific form of environmental sex determination (ESD) affects dynamics of Bonellia populations and investigate the selective advantage of ESD over the more widespread genotypic sex determination (GSD). Population dynamics of Bonellia appear rather simple and not too sensitive to parameter changes around their measured values, or to changes in distribution and sizes of inhabitable patches. Starting even from low sizes, populations soon attain equilibrium densities. Explored aspects of population dynamics indicate an advantage of ESD over GSD. Moreover, simulated invasibility experiments show that while the maternal inheritance scenario allows for fixation of GSD under some limited conditions, both the classical and proportional inheritance scenarios always lead to fixation of ESD in the population. We also show that only the ability of ESD larvae to adapt their ultimate sex both in competition for empty burrows and for mating within females gives them a competitive edge over nonadaptive response to feminising and/or masculinising signals and generally leads to fixation of ESD by small step evolution. The original hypothesis of Charnov and Bull thus needs to be refined in the sense that along with females forming an unpredictable resource for males, empty burrows are an unpredictable resource for females.     

Structural characterization of the histone variant macroH2A

macroH2A is an H2A variant with a highly unusual structural organization. It has a C-terminal domain connected to the N-terminal histone domain by a linker. Crystallographic and biochemical studies show that changes in the L1 loop in the histone fold region of macroH2A impact the structure and potentially the function of nucleosomes. The 1.6-A X-ray structure of the nonhistone region reveals an alpha/beta fold which has previously been found in a functionally diverse group of proteins. This region associates with histone deacetylases and affects the acetylation status of nucleosomes containing macroH2A. Thus, the unusual domain structure of macroH2A integrates independent functions that are instrumental in establishing a structurally and functionally unique chromatin domain.     

Liquid chromatography-mass spectrometry assay for quantitation of ifosfamide and its N-deschloroethylated metabolites in rat microsomal medium

A specific and sensitive quantitative assay has been developed using high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantitation of the antitumor drug ifosfamide (IFM) and its two metabolites, N2-deschloroethylifosfamide (N2-DCE-IFM) and N3-deschloroethylifosfamide (N3-DCE-IFM) in microsomal medium. The analytes and the internal standard (cyclophosphamide) were isolated by ethylacetate extraction from rat liver microsomes. They were analysed on a Nucleosil C18 HD column (125 mm x 4 mm, 5 microm) using a step gradient with the mobile phase (2 mM ammonium formate and methanol). The HPLC-ESI-MS method used selected ion monitoring of ions m/z 199.1 Th and m/z 261.1 Th and was validated in the concentrations ranges of 100-5000 ng/mL for IFM and 50-2500 ng/mL for its N-deschloroethylated metabolites (DCE-IFM) with good accuracy and precision (CV less than 15%). The low limits of quantitation (LLOQ) were found at 50 ng/mL for N-deschloroethylated metabolites and at 100 ng/mL for the parent drug (IFM). The method was applied for the determination of ifosfamide and its N-deschloroethylated metabolites in rat microsomal incubations.     

Economic evaluation of the effects of planting date and application rate of imidacloprid for management of cereal aphids and barley yellow dwarf in winter wheat

The effects of planting date and application rate of imidacloprid for control of Schizaphis graminum Rondani, Rhopalosiphum padi L. (Homoptera: Aphididae), and barley yellow dwarf virus (BYDV) in hard red winter wheat were studied. The first experiment was conducted from 1997 to 1999 at two locations and consisted of three planting dates and four rates of imidacloprid-treated seed. The second experiment was conducted from 2001 to 2002 in Stillwater, OK, and consisted of two varieties of hard red winter wheat seed and four rates of imidacloprid. Aphid densities, occurrence of BYDV, yield components, and final grain yield were measured, and yield differences were used to estimate the economic return obtained from using imidacloprid. In the first study, aphid populations responded to insecticide rate in the early and middle plantings, but the response was reduced in the late planting. Yields increased as insecticide rate increased but did not always result in a positive economic return. In the second study, imidacloprid seed treatments reduced aphid numbers and BYD occurrence, protected yield, and resulted in a positive economic return. The presence of aphids and BYDV lowered yield by reducing fertile head density, total kernel weight, and test weight. Whereas the application of imidacloprid seed treatments often provided positive yield protection, it did not did not consistently provide a positive economic return. A positive economic return was consistently obtained if the cereal aphid was carrying and transmitting BYDV and was more likely to occur if wheat was treated with a low rate if imidacloprid and planted in a "dual purpose" planting date window.