VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion.

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta-cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP-2 and cellubrevin, but not VAMP-1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta-cells contain secretory granules that store and secrete insulin as well as synaptic-like microvesicles carrying gamma-aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP-2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP-2 with insulin-containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta-cells. Pretreatment of streptolysin-O permeabilized insulin-secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP-2 and cellubrevin and abolished Ca(2+)-induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S-stimulated insulin secretion. Taken together, our results show that pancreatic beta-cells express VAMP-2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)-mediated insulin secretion.     

Measurement of biological information with applications from genes to landscapes

Biological diversity is quantified for reasons ranging from primer design, to bioprospecting, and community ecology. As a common index for all levels, we suggest Shannon's (S)H, already used in information theory and biodiversity of ecological communities. Since Lewontin's first use of this index to describe human genetic variation, it has been used for variation of viruses, splice-junctions, and informativeness of pedigrees. However, until now there has been no theory to predict expected values of this index under given genetic and demographic conditions. We present a new null theory for (S)H at the genetic level, and show that this index has advantages including (i) independence of measures at each hierarchical level of organization; (ii) robust estimation of genetic exchange over a wide range of conditions; (iii) ability to incorporate information on population size; and (iv) explicit relationship to standard statistical tests. Utilization of this index in conjunction with other existing indices offers powerful insights into genetic processes. Our genetic theory is also extendible to the ecological community level, and thus can aid the comparison and integration of diversity at the genetic and community levels, including the need for measures of community diversity that incorporate the genetic differentiation between species.     

Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody

Background

G-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens.

Australian Tropical and Subtropical Rain Forest Community Assembly: Phylogeny,Functional Biogeography,and Environmental Gradients

To compare community assemblage patterns in tropical northeastern and subtropical central eastern Australia across selected gradients and scales, we tested the relationship of species traits with phylogenetic structure, and niche breadth. We considered phylogenetic relationships across current‐day species in assemblages in relation to rain forest species pool sizes, and trait values along gradients including elevation and latitude. Trait values were quantified across scales for seed size, leaf area, wood density and maximum height at maturity for 1137 species and 596 assemblages using trait gradient analysis (TGA). Local assemblages of subtropical species had narrower trait ranges, and higher niche breadth values than corresponding assemblages of tropical species. Leaf size and seed size increased at low latitudes, and community phylogenetic structure was most strongly correlated with seed traits in the subtropics, reflecting dispersal and re‐colonization processes. Elevation accounted for little of the variance in community phylogenetic structure or trait variation across local and regional scales. Stable moist forest areas retained many species from ancestral rain forest lineages across a range of temporally conserved habitats; species within assemblages were less related; and rain forest assemblages had higher functional diversity, but lower niche breadth. This suggests that on average, assemblages of species in stable areas had greater trait variation and narrower distributions. Historic and recent rain forest contraction and re‐expansion can result in recolonized areas that are dominated by species that are more related (phylogenetically) than by chance, have smaller, widely dispersed seeds, and greater niche breadth (broader distributions).     

Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution

The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine.     

Site-directed mutagenesis identifies active-site residues of the light chain of botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are metalloproteases which block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such proteolysis accounts for the extreme toxicity of these neurotoxins and of their prolonged effect. The recently determined structures of BoNT/A and/B allows one to design active-site mutants to probe the role of specific residues in the proteolysis of SNARE proteins. Here we present the results of mutations of the second glutamyl residue involved in zinc coordination and of a tyrosine and a phenylalanine residues that occupy critical positions within the active site of BoNT/A. The spectroscopic properties of the purified mutants are closely similar to those of the wild-type molecule indicating the acquisition of a correct tertiary structure. Mutation of the Glu-262* nearly abolishes SNAP-25 hydrolysis as expected for a residue involved in zinc coordination. The Phe-266 and Tyr-366 mutants have reduced proteolytic activity indicating a direct participation in the proteolytic reaction, and their possible role in catalysis is discussed.     

Stable free radicals as ubiquitous components of red wines.

A stable ESR signal, centred at g = 2.0037 +/- 0.0002, characterised by a single resonance and assignable to a free radical, was found in all the bottled red wines, both commercial and experimental, that we have examined. The radical concentration was calculated to be in the range of 5-82 nM. After exposure of the wines to air for a few minutes a two fold increase of the ESR signal, followed by a slow decrease with time, was observed. The intensity of ESR signal in experimental red wines, was found to increase with the ageing of the wines and was strictly correlated to the total content of polyphenols. The formation of semiquinone radicals of polyphenols is suggested as one possible mechanism leading to the presence of stable free radicals in red wines.     

Factors influencing species selection for littoral rainforest restoration: Do environmental gradients matter?

Summary Littoral rainforest in northern New South Wales, Australia, has been severely reduced in area and is now extremely limited in extent. Factors influencing the floristics, species richness and abundance, and relationship of this coastal rainforest community type to other lowland rainforests are explored. The purpose of the study was to provide ecological information to support (i) the development of management recommendations and assist habitat expansion and restoration planning for a coastal site at Lennox Head, in northern New South Wales, and (ii) the implementation of recovery actions for an endangered ecological community and an endangered tree species. Multivariate analysis techniques were used to classify and ordinate sampled sites relative to environmental variables to provide an explanation for current floristic assemblages. Eight locations at varying distances to the coast, and representing a range of soil types, were chosen to test the influence of selected environmental variables. At the broad scale, the results show that proximity to the coast and altitude were generally correlated and represented the most influential variables; soil depth, topographic position and slope were broadly correlated but markedly less influential; disturbance was a significant but independent influence on floristics; and soil type and aspect had the least influence. The study provided insight into the ecological parameters of a range of species suited to the habitat rehabilitation and restoration project, and identified finer‐scale floristic patterns at the Lennox site that appear to reflect the influence of environmental variables. In that case, areas in closer proximity to the ocean are dominated by several littoral rainforest and disturbance‐related species, and species richness increases relative to distance from the ocean. This highlights the need to be sensitive to landscape variation, and the influence of environmental variables on plant species distributions, and population dynamics and structure, to guide final selection of appropriate plant material for littoral rainforest restoration projects.     

Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene

Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs.To determine the variants of SARS-CoV-2 circulating in the state of Nevada,specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform,which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads,without the need of culture-based amplification.High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak.We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA-dependent RNA polymerase) changed to phenylalanine(F) from proline (P),in the first reported isolate of SARS-CoV-2,Wuhan-Hu-1.This 323F variant was present at a very high frequency in Northern Nevada.Structural modeling determined this mutation in the interface domain,which is important for the association of accessory proteins required for the polymerase.In conclusion,we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations,which is important for understanding the evolution and circulation of SARS-CoV-2variants of public health importance,while it circulates in humans.