Chain collapse of an amyloidogenic intrinsically disordered protein

Natively unfolded or intrinsically disordered proteins (IDPs) are under intense scrutiny due to their involvement in both normal biological functions and abnormal protein misfolding disorders. Polypeptide chain collapse of amyloidogenic IDPs is believed to play a key role in protein misfolding, oligomerization, and aggregation leading to amyloid fibril formation, which is implicated in a number of human diseases. In this work, we used bovine κ-casein, which serves as an archetypal model protein for amyloidogenic IDPs. Using a variety of biophysical tools involving both prediction and spectroscopic techniques, we first established that monomeric κ-casein adopts a collapsed premolten-globule-like conformational ensemble under physiological conditions. Our time-resolved fluorescence and light-scattering data indicate a change in the mean hydrodynamic radius from ∼4.6 nm to ∼1.9 nm upon chain collapse. We then took the advantage of two cysteines separated by 77 amino-acid residues and covalently labeled them using thiol-reactive pyrene maleimide. This dual-labeled protein demonstrated a strong excimer formation upon renaturation from urea- and acid-denatured states under both equilibrium and kinetic conditions, providing compelling evidence of polypeptide chain collapse under physiological conditions. The implication of the IDP chain collapse in protein aggregation and amyloid formation is also discussed.     

miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha ^loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.     

GC/MS Profiling and Evaluation of Leaf Essential Oil for Bactericidal Effect and Free Radical Scavenging Activity of Plectranthus amboinicus (Lour.) Spreng Collected from Odisha,India

Plectranthus amboinicus (Lour.) Spreng, known as the Indian borage or Mexican mint, is one of the most documented species in the family Lamiaceae for its therapeutic and pharmaceutical values. It is found in the tropical and subtropical regions of the world. The leaf essential oil has immense medicinal benefits like treating illnesses of the skin and disorders like colds, asthma, constipation, headaches, coughs, and fevers. After analyzing earlier reports with regard to the quantity and quality of leaf oil yield, we discovered that the germplasm taken from Odisha is preferable to other germplasms. The objective of the present work is to evaluate the free radical scavenging activity and bactericidal effect of leaf essential oil (EO) of Plectranthus amboinicus (Lour.) Spreng collected from the state of Odisha, India. The hydro distillation technique has been used for essential oil extraction. Upon GC/MS analysis, approximately 57 compounds were identified with Carvacrol as the major compound (peak area=20.25 %), followed by p-thymol (peak area=20.17 %), o-cymene (peak area=19.41 %) and carene (peak area=15.89 %). On evaluation of free radical scavenging activity, it was recorded that the best value of inhibitory concentration, was for DPPH with IC₅₀=18.64 ppm and for H₂O₂ with IC₅₀=9.35 ppm. The EO showed efficient bactericidal effect against both gram positive (Mycobacterium smegmatis, Staphylococcus aureus, Enterococcus faecium) and gram negative (Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae) bacteria studied through well diffusion method. Fumigatory action of the essential oil was found against M. smegmatis, the model organism for tuberculosis study. Alamar Blue assay, gave a result with MIC value for M. smegmatis i. e., 0.12 μg/ml and the MBC value of 0.12 μg/ml. Hence, P. amboinicus found in Odisha can be suggested as an elite variety and should be further investigated for efficient administration in drug formulation.     

Differential effect of retinoic acid on ADP and collagen induced platelet aggregation

Retinoic acid (RA) was found to inhibit ADP induced but not collagen induced aggregation of human platelets and the differential action is related to intraplatelet Ca2+ reflux. RA was active at concentrations as low as 10(-7) M and required 20 min prior incubation with platelet suspension in order to inhibit aggregation by ADP. All the steps in ADP induced but not collagen induced platelet activation, viz. hydrolysis of phosphatidyl inositol, phosphorylation of 20, 47 and 250 kDa proteins as well as increased association of actin with Triton X-100 insoluble cytoskeletal matrix were inhibited by RA. RA when used as an agent for differentiation induction of cell progenitor is likely to affect the platelet aggregation and thereby the haemostatic process.     

Protein Kinase G Phosphorylates Mosquito-Borne Flavivirus NS5

Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to compare the phosphorylation sites of the NS5 proteins of yellow fever virus (YFV) and dengue virus (DENV), two flaviviruses transmitted by mosquitoes. Seven DENV phosphopeptides were identified, but only one conserved phosphoacceptor site (threonine 449 in DENV) was identified in both viruses. This site is predicted to be a protein kinase G (PKG) recognition site and is a strictly conserved serine/threonine phosphoacceptor site in mosquito-borne flaviviruses. In contrast, in tick-borne flaviviruses, this residue is typically a histidine. A DENV replicon engineered to have the tick-specific histidine residue at this position is replication defective. We show that DENV NS5 purified from Escherichia coli is a substrate for PKG in vitro and facilitates the autophosphorylation of PKG as seen with cellular substrates. Phosphorylation in vitro by PKG also occurs at threonine 449. Activators and inhibitors of PKG modulate DENV replication in cell culture but not replication of the tick-borne langat virus. Collectively, these data argue that PKG mediates a conserved serine/threonine phosphorylation event specifically for flaviviruses spread by mosquitoes.The flavivirus genus contains many medically important species, including dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). More than 2 billion people are at risk of infection by DENV alone, leading to an estimated 50 million cases annually, which may increase further as the range of the mosquito vector expands with urbanization (24). While disease from mosquito-borne flaviviruses is particularly common, there are other flaviviral human pathogens that exist with transmission cycles that do not involve mosquitoes. Tick-borne transmission is the other well-described route, but non-arthropod-borne routes also exist (for example, bats). It is likely that each transmission route has genetic adaptations that facilitate that route, but such changes are not yet understood (7).Serine/threonine phosphorylation is a conserved feature across all three genera of the family Flaviviridae, including the genus flavivirus (the others genera being pestivirus and hepacivirus). Among the features of Flaviviridae, the most-studied examples are the multiple phosphorylations of nonstructural protein 5A (NS5A) of hepatitis C virus, which exists in both basal (termed p56) and hyperphosphorylated (termed p58) states mediated by multiple kinases that both are necessary for and limit replication (14, 18, 23). Phosphorylation of NS5B, the RNA-dependent RNA polymerase (RdRP), has also been shown to affect replicon activity (10). In the genus flavivirus, several mosquito-borne viruses (DENV, WNV, and YFV) and at least one tick-borne encephalitis virus are known to have phosphorylated forms of nonstructural protein NS5 (2, 9, 11, 13, 19). In the genus flavivirus, NS5 is central to viral replication, as it possesses both RdRP and methyltransferase activities. DENV phosphorylation of NS5 correlates with the loss of NS5 interactions with the viral helicase NS3. A hyperphosphorylated form of NS5 was found to localize to the nucleus, away from the cytoplasmic sites of viral replication (6, 9). A nuclear localization sequence is present in DENV NS5 and is phosphorylated in vitro by host CKII, but the relationship between phosphorylation and nuclear localization has yet to be fully elucidated (17). Multiple different serine/threonine phosphorylation events likely occur in the flaviviral life cycle, potentially affecting various functions of NS5 (2), but the role of these events and identity of the kinase(s) responsible are largely unknown.In this report, we used mass spectrometry to identify serine/threonine phosphorylation sites in DENV. A single phosphoacceptor site, previously identified in YFV, is conserved specifically in the mosquito-borne flaviviruses but not the tick-borne flaviviruses. Furthermore, in vitro studies reveal that this site is phosphorylated by a cyclic-nucleotide-dependent kinase, protein kinase G (PKG), and a phosphoacceptor threonine/serine is required for replication. Taken together, these data implicate the PKG pathway in flaviviral replication for the first time and suggest a host cell pathway that could be targeted by antiviral therapy.     

Comparative Analysis of the Complete Plastid Genome Sequence of the Red Alga Gracilaria tenuistipitata var. liui Provides Insights into the Evolution of Rhodoplasts and Their Relationship to Other Plastids

We sequenced to completion the circular plastid genome of the red alga Gracilaria tenuistipitata var. liui. This is the first plastid genome sequence from the subclass Florideophycidae (Rhodophyta). The genome is composed of 183,883 bp and contains 238 predicted genes, including a single copy of the ribosomal RNA operon. Comparisons with the plastid genome of Porphyra pupurea reveal strong conservation of gene content and order, but we found major genomic rearrangements and the presence of coding regions that are specific to Gracilaria. Phylogenetic analysis of a data set of 41 concatenated proteins from 23 plastid and two cyanobacterial genomes support red algal plastid monophyly and a specific evolutionary relationship between the Florideophycidae and the Bangiales. Gracilaria maintains a surprisingly ancient gene content in its plastid genome and, together with other Rhodophyta, contains the most complete repertoire of plastid genes known in photosynthetic eukaryotes.Supplementary material () is available for this article.[Reviewing Editor: Dr. W. Ford Doolittle]     

Modifying role of dietary factors on the mutagenicity of aflatoxin B1: in vitro effect of sulphur-containing amino acids

Sulphur-containing amino acids including some derivatives have been tested for their effectiveness in suppressing the mutagenic activity of aflatoxin B1 in Salmonella typhimurium strains provided with a rat liver activation system. Cysteine and N-acetylcysteine have been found to be most effective in the 2 strains tested (TA100 and TA98). Glutathione (oxidised and reduced forms) has shown partial activity, while cystine and methionine are found to be partially effective only in strain TA100. Inhibition of mutagenicity may be due to interaction of these substances with microsomal enzymes resulting in interference with the formation of ultimate mutagenic species.     

Diversity of some insect fauna in industrial and non-industrial areas of West Bengal, India

The present work is designed to study diversity of five insect orders (viz., Hemiptera, Orthoptera, aculeate Hymenoptera, Lepidoptera and Coleoptera) in the industrial region of Haldia (India) and in non-industrial area of the same district and to evaluate the impact of industrialization on the biodiversity of those insect orders. The objective also extended to find out the possibility of existence of bioindicator, if any. Eight study sites were selected from the East part of Midnapur district covering 40 km aerial distance. Out of eight different study sites, five were distributed in and around Haldia industrial complex and three in industry-free area. During this study, a total of 120 species under 98 genera in 37 families of insects were collected. Binary data of 5 orders revealed that the species richness of Hemiptera, Orthoptera and Lepidoptera is higher in non-industrial zone in comparison to that of industrial zone. Aculeate Hymenoptera shows no particular trend whereas Coleoptera shows higher species richness in industrial areas. Results of multivariate analyses are compared with the species richness data for all the eight study sites. It is concluded that even in an apparently homogeneous ecological condition species richness may drastically change with the influence of industries. Total insect fauna decline by at least 23.33% is noticed in industrial areas. It is found that some species of lepidopteran, hemipteran and orthopteran insects are susceptible to industrial pollution and some of the members of these orders may be considered as a bioindicator group.     

Biogenic synthesis of gold nanoparticle using fractioned cellular components from eukaryotic algae and cyanobacteria

In the present investigation fractioned cellular components like intact pigment bearing thylakoids/chloroplasts, carotenoids, protein, polysaccharides were extracted from the cyanobacterium Anabaena sphaerica and green alga Chlorococcum infusionum. Each of these extracts was used separately in search for efficient reducing agents during gold nanoparticle (GNP) production in pro‐ and eukaryotic algal cell systems. The whole biomass and extracted compounds or cellular structures were exposed in 25 mg L^?1 aqueous hydrogen tetrachloroaurate solutions separately at room temperature. Isolated viable chloroplasts from C. infusionum and thylakoids from A. sphaerica were found to be able to reduce gold ions. The protein extracts of both strains were also able to synthesize GNP at 4°C. Extracted polysaccharides of the two strains responded differently. Polysaccharides from A. sphaerica showed positive response in GNP synthesis, whereas no change was observed for C. infusionum. The carotenoids extracts from both strains acted like an efficient reducing agent. Initially the reducing efficiency of these extracted components was confirmed by the appearance of purple color in biomass or in experimental media. The GNPs, synthesized within the biomass were extracted by sonication with sodium citrate. The UV–vis spectroscopy of extracted purple colored suspensions and media showed the absorption bands at approximately 530–540 nm indicating a strong positive signal of GNP synthesis. Transmission electro n microscopy determined the size and shapes of the particles. The X‐ray diffraction study of the synthesized GNP revealed that the 2θ values appeared at 38.2°, 44.5°, 64.8° and 77.8°. Amongst all, isolated thylakoids and chloroplast showed only spherical GNP production with variable size range at pH 4. Monodisperse GNPs were also synthesized by isolated thylakoids and chloroplast at pH 9. A detailed morphological change of gold treated biomass was revealed employing scanning electron microscopy. The fluorescent property of gold loaded cells was studied by fluorescence microscopy.     

Potential for Controlling Cholera Using a Ring Vaccination Strategy: Re-analysis of Data from a Cluster-Randomized Clinical Trial

IntroductionVaccinating a buffer of individuals around a case (ring vaccination) has the potential to target those who are at highest risk of infection, reducing the number of doses needed to control a disease. We explored the potential vaccine effectiveness (VE) of oral cholera vaccines (OCVs) for such a strategy.ConclusionsThese findings suggest that high-level protection can be achieved if individuals living close to cholera cases are living in a high coverage ring. Since this was an observational study including participants who had received two doses of vaccine (or placebo) in the clinical trial, further studies are needed to determine whether a ring vaccination strategy, in which vaccine is given quickly to those living close to a case, is feasible and effective.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00289224","term_id":"NCT00289224"}}NCT00289224