An Electrostatically Preferred Lateral Orientation of SNARE Complex Suggests Novel Mechanisms for Driving Membrane Fusion

Biological membrane fusion is a basic cellular process catalyzed by SNARE proteins and additional auxiliary factors. Yet, the critical mechanistic details of SNARE-catalyzed membrane fusion are poorly understood, especially during rapid synaptic transmission. Here, we systematically assessed the electrostatic forces between SNARE complex, auxiliary proteins and fusing membranes by the nonlinear Poisson-Boltzmann equation using explicit models of membranes and proteins. We found that a previously unrecognized, structurally preferred and energetically highly favorable lateral orientation exists for the SNARE complex between fusing membranes. This preferred orientation immediately suggests a novel and simple synaptotagmin-dependent mechanistic trigger of membrane fusion. Moreover, electrostatic interactions between membranes, SNARE complex, and auxiliary proteins appear to orchestrate a series of membrane curvature events that set the stage for rapid synaptic vesicle fusion. Together, our electrostatic analyses of SNAREs and their regulatory factors suggest unexpected and potentially novel mechanisms for eukaryotic membrane fusion proteins.     

Development and Evaluation of a Novel Delivery System Containing Phytophospholipid Complex for Skin Aging

Citrus auranticum and Glycyrrhiza glabra are rich in anti-oxidant polyphenols helpful in prevention of skin aging. Polyphenols have high polarity and lower skin penetration resulting in lower cutaneous delivery. The present work is attempted to develop a novel polyherbal phospholipid complex cream to improve cutaneous delivery of polyphenols for sustained anti-oxidant action. Phytochemical and in vitro anti-oxidant evaluation was done on methanolic extracts of orange peel and liquorice powder. Total phenolic content, total flavonoid content, and anti-oxidant assays were done on different ratios of orange peel and liquorice extract. Ratio 1:2 gave highest total phenolic content (TPC) (530.00?±?1.56 mg gallic acid equivalent (GAE)?g^?1 extract), total flavonoid content (TFC) (246.25?±?1.03 mg rutin equivalent (RUE)?g^?1 extract), 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity (87.99?±?0.64%), and H₂O₂ scavenging activity (72.47?±?0.86%) and hence was used for formulation. Solvent evaporation method using methanol with 1:1 extract to phospholipid ratio was found to have entrapment efficiency of 93.22?±?0.26%. Evaluation parameters like scanning electron microscopy (SEM), Fourier transform infrared spectrophotometry (FT-IR), and differential scanning calorimetry (DSC) confirmed formation of complex. The complex was formulated as oil-in-water cream and evaluated for various parameters. The optimized cream containing 1% complex was non-irritant and was found to be stable for 3-month period under conditions of stability study. Ex vivo diffusion studies showed that extract phospholipid complex cream had better retention of polyphenols in the skin when compared to conventional extract cream giving prolonged and stronger topical action. The cream had an anti-elastase activity of 28.02?±?0.95% at concentration of 3000 μg ml^?1 (w/v). Thus, the developed safe and stable polyherbal phytophospholipid complex cream exhibited good potential as anti-aging cosmeceutical.     

Repetitive,Marker-Free,Site-Specific Integration as a Novel Tool for Multiple Chromosomal Integration of DNA

We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attB_min), two mutated loxP sequences (lox66 and lox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection of loxP recombination on the pKV6 vector. When transformed into L. lactis expressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked by attL and attR hybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate in L. lactis, loxP recombinants can be selected for by using 5-fluoroorotic acid. The introduced attB_min site can subsequently be used for a second round of integration. To examine if attP recombination was specific to the attB site, integration was performed in strains containing the attB, attL, and attR sites or the attL and attR sites only. Only attP-attB recombination was observed when all three sites were present. In the absence of the attB site, a low frequency of attP-attL recombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABR and xylT) from L. lactis strain KF147 were integrated into the chromosome of L. lactis strain MG1363 in two steps.     

A Genome-Wide Association Study Suggests Novel Loci Associated with a Schizophrenia-Related Brain-Based Phenotype

Patients with schizophrenia and their siblings typically show subtle changes of brain structures, such as a reduction of hippocampal volume. Hippocampal volume is heritable, may explain a variety of cognitive symptoms of schizophrenia and is thus considered an intermediate phenotype for this mental illness. The aim of our analyses was to identify single-nucleotide polymorphisms (SNP) related to hippocampal volume without making prior assumptions about possible candidate genes. In this study, we combined genetics, imaging and neuropsychological data obtained from the Mind Clinical Imaging Consortium study of schizophrenia (n = 328). A total of 743,591 SNPs were tested for association with hippocampal volume in a genome-wide association study. Gene expression profiles of human hippocampal tissue were investigated for gene regions of significantly associated SNPs. None of the genetic markers reached genome-wide significance. However, six highly correlated SNPs (rs4808611, rs35686037, rs12982178, rs1042178, rs10406920, rs8170) on chromosome 19p13.11, located within or in close proximity to the genes NR2F6, USHBP1, and BABAM1, as well as four SNPs in three other genomic regions (chromosome 1, 2 and 10) had p-values between 6.75×10⁻⁶ and 8.3×10⁻⁷. Using existing data of a very recently published GWAS of hippocampal volume and additional data of a multicentre study in a large cohort of adolescents of European ancestry, we found supporting evidence for our results. Furthermore, allelic differences in rs4808611 and rs8170 were highly associated with differential mRNA expression in the cis-acting region. Associations with memory functioning indicate a possible functional importance of the identified risk variants. Our findings provide new insights into the genetic architecture of a brain structure closely linked to schizophrenia. In silico replication, mRNA expression and cognitive data provide additional support for the relevance of our findings. Identification of causal variants and their functional effects may unveil yet unknown players in the neurodevelopment and the pathogenesis of neuropsychiatric disorders.     

Integrative and Sequence Characteristics of a Novel Genetic Element,ICE6013, in Staphylococcus aureus

A survey of chromosomal variation in the ST239 clonal group of methicillin-resistant Staphylococcus aureus (MRSA) revealed a novel genetic element, ICE6013. The element is 13,354 bp in length, excluding a 6,551-bp Tn552 insertion. ICE6013 is flanked by 3-bp direct repeats and is demarcated by 8-bp imperfect inverted repeats. The element was present in 6 of 15 genome-sequenced S. aureus strains, and it was detected using genetic markers in 19 of 44 diverse MRSA and methicillin-susceptible strains and in all 111 ST239 strains tested. Low integration site specificity was discerned. Multiple chromosomal copies and the presence of extrachromosomal circular forms of ICE6013 were detected in various strains. The circular forms included 3-bp coupling sequences, located between the 8-bp ends of the element, that corresponded to the 3-bp direct repeats flanking the chromosomal forms. ICE6013 is predicted to encode 15 open reading frames, including an IS30-like DDE transposase in place of a Tyr/Ser recombinase and homologs of gram-positive bacterial conjugation components. Further sequence analyses indicated that ICE6013 is more closely related to ICEBs1 from Bacillus subtilis than to the only other potential integrative conjugative element known from S. aureus, Tn5801. Evidence of recombination between ICE6013 elements is also presented. In summary, ICE6013 is the first member of a new family of active, integrative genetic elements that are widely dispersed within S. aureus strains.ST239 is a globally distributed clonal group of methicillin-resistant Staphylococcus aureus (MRSA). Currently, ST239 is a major cause of MRSA infections in Asian hospitals (5, 18, 25, 37, 45, 64, 74). Pulsed-field gel electrophoresis has detected extensive chromosomal variation in local ST239 populations (3, 24, 52, 72). As ST239 has geographically spread and diversified, its variants have been given more than a dozen different names (20, 22, 24, 25, 49, 52, 61, 67, 68, 73), which reflects their clinical significance in various locales. The molecular basis for the ecological success of ST239 is unclear, but virulence-associated traits such as enhanced biofilm development and epidemiological characteristics such as a propensity to cause device-associated bacteremia and pulmonary infections have been highlighted (3, 19, 27, 54).Multilocus genetic investigations of the ST239 chromosome revealed that it is a hybrid with estimated parental contributions of approximately 20% and 80% from distantly related ST30- and ST8-like parents, respectively (58). Unusual for naturally isolated bacteria was the finding that these parental contributions were large chromosomal replacements rather than a patchwork of localized recombinations. It was postulated that conjugation might be responsible for the natural transfer of hundreds of kilobases of contiguous chromosomal DNA that resulted in ST239 (58). Recent genomic investigations have presented evidence that large chromosomal replacements also occur within Streptococcus agalactiae strains and that they can be mimicked with laboratory conjugation experiments (12). Importantly, conjugative transfer frequencies in S. agalactiae were found to be highest near three genomic islands (12), two of which were identified as being integrative conjugative elements (ICEs) (13).ICEs and conjugative transposons are synonyms and refer to genetic elements that are maintained by integration into a replicon and are transmitted by self-encoded conjugation functions (56). ICEs abound in the genomes of S. agalactiae (11), but only one potential ICE has been identified in staphylococci to date: Tn5801 was discovered through the genomic sequencing of S. aureus strain Mu50 (46). Tn5801 is most similar to a truncated genetic element, CW459tet(M), from Clostridium perfringens (57). Both Tn5801 and CW459tet(M) have Tyr recombinases, regulatory genes, and tetM modules that are similar to those of the prototypical gram-positive conjugative transposon, Tn916. Moreover, both Tn5801 and CW459tet(M) integrate into the same locus, guaA, at a nearly identical 11-bp sequence. Although the conjugative transfer module of CW459tet(M) is deleted (57), the conjugative transfer module of Tn5801 is similar to that of Tn916.We suspected that ST239 strains might carry novel accessory genes that contribute to their chromosomal variation and ecological success. To explore this possibility, we conducted a survey of chromosomal variation in ST239 using a PCR scanning approach. We report the discovery and partial characterization of a novel genetic element, ICE6013, that resulted from the survey.     

Characterization of a Novel Integrative Element, ICESt1, in the Lactic Acid Bacterium Streptococcus thermophilus

The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3′ end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276 and Tn5252. The integrase was found to be involved in a site-specific excision of a circular form. ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916. Therefore, ICESt1 could be or could be derived from an integrative conjugative element.     

The Role of Citrullinated Proteins Suggests a Novel Mechanism in the Pathogenesis of Multiple Sclerosis

The pathogenesis of MS is unknown. In our studies, we have demonstrated an important role for citrullinated myelin basic protein (MBP). The accompanying loss of positive charge compromises the ability of MBP to interact with the lipid bilayer. The conversion of arginine to citrulline in brain is carried out by an enzyme peptidyl arginine deiminase (PAD) 2. The amount of PAD 2 in brain was increased in MS normal-appearing white matter. The mechanism responsible for this increase involved hypomethylation of the promoter region in the PAD 2 gene in MS, but no change (compared to normal) was found in thymus tissue DNA from the same MS patients. In addition, no change was observed in other neurological diseases, including Alzheimer’s, Parkinson’s, and Huntington’s. We propose that citrullinated MBP, resulting from elevated levels of PAD 2 represents an important biochemical pathway in the pathogenesis of MS. Special issue dedicated to Anthony Campagnoni.     

Structure of a VP1-VP3 Complex Suggests How Birnaviruses Package the VP1 Polymerase

Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae, infects young salmon, with a severe impact on the commercial sea farming industry. Of the five mature proteins encoded by the IPNV genome, the multifunctional VP3 has an essential role in morphogenesis; interacting with the capsid protein VP2, the viral double-stranded RNA (dsRNA) genome and the RNA-dependent RNA polymerase VP1. Here we investigate one of these VP3 functions and present the crystal structure of the C-terminal 12 residues of VP3 bound to the VP1 polymerase. This interaction, visualized for the first time, reveals the precise molecular determinants used by VP3 to bind the polymerase. Competition binding studies confirm that this region of VP3 is necessary and sufficient for VP1 binding, while biochemical experiments show that VP3 attachment has no effect on polymerase activity. These results indicate how VP3 recruits the polymerase into birnavirus capsids during morphogenesis.     

In Silico Analysis of Phosphoproteome Data Suggests a Rich-get-richer Process of Phosphosite Accumulation over Evolution

Recent phosphoproteome analyses using mass spectrometry-based technologies have provided new insights into the extensive presence of protein phosphorylation in various species and have raised the interesting question of how this protein modification was gained evolutionarily on such a large scale. We investigated this issue by using human and mouse phosphoproteome data. We initially found that phosphoproteins followed a power-law distribution with regard to their number of phosphosites: most of the proteins included only a few phosphosites, but some included dozens of phosphosites. The power-law distribution, unlike more commonly observed distributions such as normal and log-normal distributions, is considered by the field of complex systems science to be produced by a specific rich-get-richer process called preferential attachment growth. Therefore, we explored the factors that may have promoted the rich-get-richer process during phosphosite evolution. We conducted a bioinformatics analysis to evaluate the relationship of amino acid sequences of phosphoproteins with the positions of phosphosites and found an overconcentration of phosphosites in specific regions of protein surfaces and implications that in many phosphoproteins these clusters of phosphosites are activated simultaneously. Multiple phosphosites concentrated in limited spaces on phosphoprotein surfaces may therefore function biologically as cooperative modules that are resistant to selective pressures during phosphoprotein evolution. We therefore proposed a hypothetical model by which the modularization of multiple phosphosites has been resistant to natural selection and has driven the rich-get-richer process of the evolutionary growth of phosphosite numbers.Protein phosphorylation is an important and ubiquitous post-translational modification that regulates a variety of biological processes in various organisms (1–4). Reversible phosphorylations of serine, threonine, and tyrosine residues are critical steps in the control of signal transduction pathways (1–4). Recent advances in MS-based technologies and phosphopeptide enrichment methods have allowed high throughput and large scale in vivo phosphosite mapping for a wide variety of organisms such as human (5–8), mouse (9), yeast (10–12), fly (13,14), bacteria (15,16), and plants (17–19). Moreover information on several hundred to several thousand phosphosites from each study has been gathered in public databases such as Phospho.ELM (20), PhosphoSitePlus, PHOSIDA (21), and UniProt (22). However, the total number of phosphosites and most of their biological functions are still unknown. Similarly only about 10% of the estimated 500–600 human kinases target known phosphosite consensus sequences within their substrate proteins (23). Although the tyrosine phosphoproteome in Arabidopsis was recently published (24), the corresponding tyrosine kinases have not been identified because of the lack of known consensus sequences activated by tyrosine kinases.Computational data-mining approaches have been required to extract information from the large amount of accumulated phosphosite data obtained from experimental approaches. These approaches have also been used to add more meaningful information about each of the phosphosites to understand the proteome-wide protein phosphorylation in various organisms. One of the most useful strategies of computational data mining is to identify phosphorylated sequence motifs by extracting consensus sequences from the sets of amino acid sequences clustered around phosphorylated residues (25). A number of kinases and their corresponding recognition substrate motifs have been successfully identified by the experimental approach of incubating each target kinase with a combinatorial substrate peptide library and ATP, and these data are registered in various databases, including the Human Protein Reference Database (HPRD)¹ (26). With this knowledge of documented kinases and their related sequence motifs, we can use computational biology techniques to discover additional phosphorylated motifs in the numerous substrates shown in phosphoproteomics studies to be biologically phosphorylated. This has allowed us to reconstruct the kinome on a large scale (27–29).A comparative study of phosphoproteome data in multiple species has revealed that a wide range of phosphoproteins are relatively well conserved relative to non-phosphoproteins, and similarly many phosphoserine (Ser(P)), phosphothreonine (Thr(P)), and phosphotyrosine (Tyr(P)) phosphosites are well conserved compared with non-phosphorylated sites (21). Under natural selection, the emergence of phosphoproteins and the gain and loss of phosphosites should have changed the regulation of many intracellular systems, such as kinetic pathways, subcellular protein localization, and protein interactions and stabilization. This triggered our interest in the evolution of phosphoproteins and their phosphosites.In this study, we combined statistical physics and computational biology to investigate the role of selective pressure in the evolution of phosphoproteins and to create a model of the evolutionary gain of phosphosites. First using the human and mouse phosphoproteome data registered in public databases, we discovered that the number of phosphosites in each phosphoprotein follows a power-law distribution, which has been shown in complex systems science and statistical physics to emerge through a specific rich-get-richer process called preferential attachment growth (30–32). We therefore hypothesized that phosphoproteins may have evolved through a rich-get-richer process, gaining new phosphosites according to a probability density proportional to their current number of phosphosites. Starting from this hypothesis, we then explored how this particular evolutionary pattern may have arisen during natural selection and suggested that sets of phosphosites localized in limited spaces on protein surfaces may function as cooperative modules that are resistant to selective pressures. Therefore, to explain phosphosite evolution, we proposed a model in which the evolutionary gain of phosphosites follows a rich-get-richer process and evolution is promoted by the development of cooperative functional modules on protein surfaces.     

Detection and Characterization of a Novel Norovirus in Bats,China

正Dear Editor,Noroviruses(No Vs)are second only to the rotaviruses as etiologic agents of acute fulminant gastroenteritis in infants and young children worldwide,with an estimated 200,000deaths per year in children younger than 5 years of age in developing countries(Patel et al.2008).No Vs are classified within the genus Norovirus of the family Caliciviridae with Norwalk virus as its prototype member(ICTV 2017).The virions are small(38–40 nm in diameter)nonenveloped,with an icosahedral capsidanda linear,positive-     

Detection of a Target Taste in a Complex Masker

Detection thresholds for sodium chloride were compared in aqueoussolution, in mixture with a sucrose masker, in mixture witha citric acid masker, and in mixture with both of these maskerstogether. Separately the two maskers raised the threshold ofsodium chloride by three to four times, and together by overnine times, a result consistent with independence (additivity)of the two masking effects. To achieve comparable masking witheither sucrose alone or with citric acid alone would requireincreasing their masking concentrations by about ten times.Hence multiple masking can be a far more efficient means ofconcealing a taste, whether an unpleasant one (e.g. the bittertaste of medicine) or a pleasant one (e.g. a salty or sweetcondiment). Multiple masking has dietary and culinary significance,especially for middle aged and elderly persons concerned aboutsalt intake, because their thresholds for NaCl, whether withor without maskers, are typically two or three times higherthan those of youthful persons. Chem. Senses 22: 529–534,1997.     

Nest Site Selection by Kentish Plover Suggests a Trade-Off between Nest-Crypsis and Predator Detection Strategies

Predation is one of the main causes of adult mortality and breeding failure for ground-nesting birds. Micro-habitat structure around nests plays a critical role in minimizing predation risk. Plovers nest in sites with little vegetation cover to maximize the incubating adult visibility, but many studies suggest a trade-off between nest-crypsis and predator detection strategies. However, this trade-off has not been explored in detail because methods used so far do not allow estimating the visibility with regards to critical factors such as slope or plant permeability to vision. Here, we tested the hypothesis that Kentish plovers select exposed sites according to a predator detection strategy, and the hypothesis that more concealed nests survive longer according to a crypsis strategy. To this end, we obtained an accurate estimation of the incubating adult''s field of vision through a custom built inverted periscope. Our results showed that plovers selected nest sites with higher visibility than control points randomly selected with regards to humans and dogs, although nests located in sites with higher vegetation cover survived longer. In addition, the flushing distance (i.e., the distance at which incubating adults leave the nest when they detect a potential predator) decreased with vegetation cover. Consequently, the advantages of concealing the nest were limited by the ability to detect predators, thus indirectly supporting the existence of the trade-off between crypsis and predator detection. Finally, human disturbance also constrained nest choice, forcing plovers to move to inland sites that were less suitable because of higher vegetation cover, and modulated flushing behavior, since plovers that were habituated to humans left their nests closer to potential predators. This constraint on the width of suitable breeding habitat is particularly relevant for the conservation of Kentish Plover in sand beaches, especially under the current context of coastal regression and increase of recreational activities.     

Detection of a Novel Bovine Lymphotropic Herpesvirus

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.