Transmissibility of intra-host hepatitis C virus variants

Background

Intra-host hepatitis C virus (HCV) populations are genetically heterogeneous and organized in subpopulations. With the exception of blood transfusions, transmission of HCV occurs via a small number of genetic variants, the effect of which is frequently described as a bottleneck. Stochasticity of transmission associated with the bottleneck is usually used to explain genetic differences among HCV populations identified in the source and recipient cases, which may be further exacerbated by intra-host HCV evolution and differential biological capacity of HCV variants to successfully establish a population in a new host.

Results

Transmissibility was formulated as a property that can be measured from experimental Ultra-Deep Sequencing (UDS) data. The UDS data were obtained from one large hepatitis C outbreak involving an epidemiologically defined source and 18 recipient cases. k-Step networks of HCV variants were constructed and used to identify a potential association between transmissibility and network centrality of individual HCV variants from the source. An additional dataset obtained from nine other HCV outbreaks with known directionality of transmission was used for validation.Transmissibility was not found to be dependent on high frequency of variants in the source, supporting the earlier observations of transmission of minority variants. Among all tested measures of centrality, the highest correlation of transmissibility was found with Hamming centrality (r?=?0.720; p?=?1.57 E-71). Correlation between genetic distances and differences in transmissibility among HCV variants from the source was found to be 0.3276 (Mantel Test, p?=?9.99 E-5), indicating association between genetic proximity and transmissibility. A strong correlation ranging from 0.565–0.947 was observed between Hamming centrality and transmissibility in 7 of the 9 additional transmission clusters (p?<?0.05).

Conclusions

Transmission is not an exclusively stochastic process. Transmissibility, as formally measured in this study, is associated with certain biological properties that also define location of variants in the genetic space occupied by the HCV strain from the source. The measure may also be applicable to other highly heterogeneous viruses. Besides improving accuracy of outbreak investigations, this finding helps with the understanding of molecular mechanisms contributing to establishment of chronic HCV infection.

     

Rapid detection of ESBL-producing Klebsiella pneumoniae in blood cultures by fluorescent in-situ hybridization

Multi-resistant Enterobacteriaceae pose a serious threat of hospital acquired infections and their rapid identification is important for better clinical outcome. This study describes the rapid identification of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae of the sulphydryl variable-type by fluorescent in-situ hybridization. The method which rapidly identifies the target genes within 1 h could be a potentially rapid bacterial diagnostic tool.     

Acidogenic fermentation of proteinaceous solid waste and characterization of different bioconversion stages and extracellular products

The purpose of this study was to investigate hydrolysis of animal fleshing (ANFL), a predominant tannery solid waste and to characterize the acetogenic fermentation products of anaerobic digestion. The acidogenic digestibility of the tannery solid wastes were evaluated up to 120 h using batch anaerobic digestion tests performed under mesophilic condition at 37 degrees C. The degradation of ANFL starts with non-fibrillar proteins and proceeds with fibrillar proteins. The release of aliphatic amino acid in the early stages of hydrolysis (24 h) and followed by aromatic amino acids (24-72 h) were evidenced by HPLC analysis. The maximum production of propionic and valeric acid were observed in 72 h followed by rapid increase in acetic acid in 96 h using GC-MS. Breakdown of ANFL and formations of other metabolites were evidenced by FT-IR and (1)H-NMR spectroscopy.     

Kinetic and equilibrium studies on the biosorption of reactive black 5 dye by Aspergillus foetidus

An isolated fungus, Aspergillus foetidus had the ability to decolourize growth unsupportive medium containing 100 mg L(-1) of reactive black 5 (RB5) dye with >99% efficiency at acidic pH (2-3). Pre-treatment of fungal biomass by autoclaving or exposure to 0.1M sodium hydroxide facilitated more efficient uptake of dye as compared to untreated fungal biomass. Pre-equilibrium biosorption of RB5 dye onto fungus under different temperatures followed pseudo-second-order kinetic model with high degree of correlation coefficients (R(2)>0.99). Biosorption isotherm data fitted better into Freundlich model for lower concentrations of dye probably suggesting the heterogeneous nature of sorption process. Based on the Langmuir isotherm plots the maximum biosorption capacity (Q(0)) value was calculated to be 106 mg g(-1) at 50 degrees C for fungal biomass pre-treated with 0.1M NaOH. Thermodynamic studies revealed that the biosorption process was favourable, spontaneous and endothermic in nature. Recovery of both adsorbate (dye) and adsorbent (fungal biomass) was possible using sodium hydroxide. Recovered fungal biomass could be recycled number of times following desorption of dye using 0.1M NaOH. Fungal biomass pre-treated with NaOH was efficient in decolourizing solution containing mixture of dyes as well as composite raw industrial effluent generated from leather, pharmaceutical and dye manufacturing company.     

Purification of extracellular acid protease and analysis of fermentation metabolites by Synergistes sp. utilizing proteinaceous solid waste from tanneries

The untanned proteinaceous tannery solid waste, animal fleshing (ANFL), was used as a substrate for acid protease production by Synergistes sp. The strain was isolated from an anaerobic digester used for the treatment of tannery solid waste and was selected for its enhanced protease production at activity 350-420 U/ml. The optimum pH was in the acidic range of 5.5-6.5 and optimum temperature was in mesophilic range of 25-35 degrees C. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the zymogram analyses of the purified protein indicated an estimated molecular mass of 60 kDa. This protease could be classified as aspartic protease based on its inhibition by aspartate type protease inhibitor pepstatin and on non-inhibition by 1,10-phenanthroline, EDTA, EGTA and phenylmethylsulfonyl fluoride. The degradation of ANFL was confirmed by Gas Chromatography-Mass Spectroscopy (GC-MS), Proton Nuclear Magnetic Resonance Spectroscopy (H1 NMR) and Scanning Electron Microscopy (SEM) analyses. In this study we found that the activity of acid protease depended on factors such as calcium concentration, pH and temperature. Based on these lines of evidence, we postulate that this protease is a highly catalytic novel protease of its type.     

Removal of tannin from cross-linked and open chain polymeric tannin substrates using heme peroxidases of Phanerochaete chrysosporium

Removal of tannin from different tannin substrates using heme peroxidases of Phanerochaete chrysosporium was studied. Complete removal of tannin components in spent tan liquor was observed after 24 h of incubation with peroxidases. Tannin in aqueous media containing tannic acid and condensed tannin substrates were removed by 65.70ǂ.97 and 52.43ǂ.83%. Chemical oxygen demand was reduced by 24.38ǂ.73, 33.22ǂ.20, and 58.94ǂ.07%, respectively, in spent tan liquor, tannic acid and condensed tannin substrates containing aqueous media.     

Reactivity of 3-HBA-6-hydroxylase with diethylpyrocarbonate and N-bromosuccinimide: effect of chemical modifications on kinetic and spectral properties of the enzyme

The rapid inactivation of 3-HBA-6-hydroxylase by 100 microM diethylpyrocarbonate or 40 microM N-bromosuccinimide and protection offered by the substrate, 3-hydroxybenzoate, against these chemical modifications implicate the involvement of histidine and tryptophan in the catalytic activity of the enzyme. Inactivation of the enzyme by diethylpyrocarbonate followed pseudo-first-order kinetics, and an "n" value of 1.3 was obtained. Inactivation of the enzyme by N-bromosuccinimide was instantaneous and failed to follow pseudo-first-order kinetics. Distinct and incremental changes in the UV absorption, emission fluorescence, and near UV-CD spectra of the enzyme upon its titration with increasing concentrations of diethylpyrocarbonate or N-bromosuccinimide may be ascribed to modification and/or changes in the microenvironment of aromatic amino acid residue(s) such as tryptophan in the enzyme.     

Soil microbial community structure and enzymatic activity responses to nitrogen management and landscape positions in switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is usually grown on marginal land for biofuel system, in which nitrogen (N) is an essential management practice, and landscape position is a key topographical factor in impacting the production. However, limited information is available regarding how the N application and landscape positions affect soil microbial communities and enzyme activities under switchgrass. Thus, the specific objective of this study was to evaluate the responses of N rate (high, 112 kg N/ha; medium, 56 kg N/ha; and low, 0 kg N/ha) and landscape positions (shoulder and footslope) on soil biological health under switchgrass field. Data showed that N addition significantly influenced carbon and N fractions. The hot water‐soluble organic carbon (HWC) and nitrogen (HWN) fractions were significantly higher at footslope position than the shoulder position. The amount of total phospholipid fatty acid (PLFA), total bacterial, actinomycetes, gram‐negative and gram‐positive bacteria, total fungi, arbuscular mycorrhizal (AM) fungi, and saprophytes PLFAs were highest with medium and high N rates and footslope position. The N addition increased total PLFAs in N fertilizer treatments, viz. medium (5,946 ng PLFA‐C/g soil) and high N rates (5,871 ng PLFA‐C/g soil). Microbial biomass carbon and nitrogen and enzyme activities (urease, β‐glucosidase, acid phosphatase and arylsulfatase) were significantly enhanced by N fertilization (medium and high N rates) compared to control (low N rates) under footslope position. The urease activity under medium (36.3 µmol N‐NH₄⁺ g^?1 soil hr^?1) and high N rates (31.4 µmol N‐NH₄⁺ g^?1 soil hr^?1) was 42.9% and 23.6% higher than low N rates, respectively. This study suggests that the application of medium N rate in footslope position to switchgrass can enhance the soil biological properties and hence can protect the environment from the excessive use of N fertilizer.     

The O‐methyltransferase gene MdoOMT1 is required for biosynthesis of methylated phenylpropenes in ripe apple fruit

Phenylpropenes, such as eugenol and trans‐anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala (RG, estragole producer) × Granny Smith (GS, non‐producer) apple cross. Two quantitative trait loci (QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group (LG) 1 and LG2 were identified that co‐located with seven candidate genes for phenylpropene O‐methyltransferases (MdoOMT1–7). Of these genes, only expression of MdoOMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over‐expression in tobacco showed that MdoOMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles (MdoOMT1a, MdoOMT1b) whilst GS appeared to be homozygous for MdoOMT1b. MdoOMT1a showed a higher affinity and catalytic efficiency towards chavicol than MdoOMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced MdoOMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that MdoOMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma.