Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.Hepatitis C virus (HCV) infection is a major global health issue leading to persistent viral infection in the majority of those infected and is associated with progressive liver disease, cirrhosis, and hepatocellular carcinoma. Six major genotypes of HCV have been described that have evolved in geographically distinct regions and that share approximately. 80% nucleotide homology with one another. HCV viral genotypes have been further classified into subtypes (25). HCV subtype 3a infection is now the most common subtype in the United Kingdom (11), although it is globally distributed and frequently associated with intravenous drug use.The classification of HCV viral strains by genotype and subtype has proven informative not only in terms of the epidemic and evolutionary history of the virus but also in terms of clinical outcomes. In particular, the response rates to current gold standard therapy (9) and the prevalence of hepatic steatosis (20) are significantly higher for subtype 3a than for genotype 1 infections. The reasons for this are not understood but must relate to viral genetic and phenotypic differences between strains, or to differences in the ability of hosts to exert an effective immune response against particular viral sequences, or to a combination of both factors.To date, detailed assessment of the HCV genome has largely focused on HCV genotype 1. Indeed, only a few full-length HCV subtype 3a viral sequences are currently published and available within the major HCV databases (Los Alamos; http://hcv.lanl.gov/components/hcv-db/combined_search/searchi.html and euHCVdb; http://euhcvdb.ibcp.fr/euHCVdb/) (16).To characterize HCV subtype 3a in detail, we performed whole-genome analysis of a cohort of patients with persistent HCV subtype 3a infection. We subsequently focus on the highly variable regions observed in the envelope protein E2 in both acute and chronic infection, since it was apparent that these regions were not restricted to the well-documented hypervariable region 1 (HVR1) that is found at the 5′ end of E2 in all HCV genotypes.Viral genomic variability can be assessed at a number of different levels; first, intergenotypic variability may arise in genomic regions that are conserved within the same subtype but are distinct between subtypes. Second, there is intragenotypic variability, which may be defined as regions of viral variability within the same genotype or subtype. Finally, intrahost variability is where viral genomic variability occurs within the same viral subtype and also the same host when individual clonal sequences are assessed. Although intergenotypic variability may simply be a feature of the existence of geographically distinct HCV subtypes, intragenotypic and intrahost variability may reflect viral regions subject to specific selection pressures, with important functional implications.We observed two distinct regions of intrahost and intragenotypic hypervariability within genotype 3a envelope 2 (E2)—in addition to the previously described HVR1—that we have named HVR495 and HVR575. We show that these regions are subject to positive selection pressure, sometimes very early in acute infection. Although HVR575 has been previously recognized as a site of intergenotypic variation (18), the identification of this region as a hypervariable site within genotype 3a and as a site under early selection pressure leading to variability within the same host has not been previously described.     

A colorimetric method to quantify endo-polygalacturonase activity

We report a new colorimetric assay to quantify endo-polygalacturonase activity, which hydrolyzes polygalacturonic acid to produce smaller chains of galacturonate. Some of the reported polygalacturonase assays measure the activity by detecting the appearance of reducing ends such as the Somogyi-Nelson method. As a result of being general towards reducing groups, the Somogyi-Nelson method is not appropriate when studying polygalacturonase and polygalacturonase inhibitors in plant crude extracts, which often have a strong reducing power. Ruthenium Red is an inorganic dye that binds polygalacturonic acid and causes its precipitation. In the presence of polygalacturonase, polygalacturonic acid is hydrolyzed bringing about a corresponding gain in soluble Ruthenium Red. The described assay utilizes Ruthenium Red as the detection reagent which has been used previously in plate-based assays but not in liquid medium reactions. The new method measures the disappearance of the substrate polygalacturonic acid and is compared to the Somogyi-Nelson assay. The experimental results using lemon peel, a fern fronds and castor leaf crude extracts demonstrate that the new method provides a way to the quickly screening of polygalacturonase activity and polygalacturonase inhibitors in plant crude extracts containing high amounts of reducing power. On the other hand, the Ruthenium Red assay is not able to determine the activity of an exo-polygalacturonase as initial velocity and thus would allow the differentiation between endo- and exo-polygalacturonase activities.     

A synthesis of methane emissions from 71 northern,temperate, and subtropical wetlands

Wetlands are the largest natural source of atmospheric methane. Here, we assess controls on methane flux using a database of approximately 19 000 instantaneous measurements from 71 wetland sites located across subtropical, temperate, and northern high latitude regions. Our analyses confirm general controls on wetland methane emissions from soil temperature, water table, and vegetation, but also show that these relationships are modified depending on wetland type (bog, fen, or swamp), region (subarctic to temperate), and disturbance. Fen methane flux was more sensitive to vegetation and less sensitive to temperature than bog or swamp fluxes. The optimal water table for methane flux was consistently below the peat surface in bogs, close to the peat surface in poor fens, and above the peat surface in rich fens. However, the largest flux in bogs occurred when dry 30‐day averaged antecedent conditions were followed by wet conditions, while in fens and swamps, the largest flux occurred when both 30‐day averaged antecedent and current conditions were wet. Drained wetlands exhibited distinct characteristics, e.g. the absence of large flux following wet and warm conditions, suggesting that the same functional relationships between methane flux and environmental conditions cannot be used across pristine and disturbed wetlands. Together, our results suggest that water table and temperature are dominant controls on methane flux in pristine bogs and swamps, while other processes, such as vascular transport in pristine fens, have the potential to partially override the effect of these controls in other wetland types. Because wetland types vary in methane emissions and have distinct controls, these ecosystems need to be considered separately to yield reliable estimates of global wetland methane release.     

Changing climate sensitivity of black spruce (Picea mariana Mill.) in a peatland–forest landscape in Interior Alaska

Dendroclimatological research is often based on the assumption that the relationship between tree growth and climate is not variable over time. Here we test this assumption by exploring if climate sensitivity of Picea mariana (Mill.) trees growing in open-stand lowland forest and on top of a neighboring peatland in Interior Alaska is stable or changing over time. Climate–growth correlations at the study sites are strongly dependent on microtopography and vary substantially over time. Trees growing in the open forest site generally display stronger climate–growth correlations, especially significantly negative correlations with late summer temperatures (July, August) starting in the period 1920–1970. Trees growing on the peatland site are less climate sensitive, but display positive correlations between annual growth and temperature of October and December in the early 20th century, while in the late 20th century, significant negative correlations exist with January and February temperatures. This study, thus, demonstrates a transient climate–growth response for P. mariana (Mill.) on two sites typical for lowland Interior Alaska. However, due to multiple possible explanations (e.g. changing climate, coupled with aging trees and a growing peatland surface) it is not possible at this time to pinpoint the exact cause for these changes in the climate–growth relationships.     

Emergence of Resistance to Fluconazole in <Emphasis Type="Italic">Candida albicans</Emphasis> Isolated From Vaginal Discharge

Purpose of Review

To provide information about the emergence of fluconazole resistance in Candida albicans isolated from vaginal discharge, in a global context, and to update the in vitro susceptibility profile of this species from Argentina.

Recent Findings

Vulvovaginal candidiasis is the second most common vaginal infection after vaginal bacteriosis. C. albicans remains the prevalent etiological yeast species, and despite antifungal treatment, the rate of recurrence remains high, which may be associated to antifungal resistance.

Summary

Data here presented were obtained from the study of C. albicans strains isolated from patients with clinical signs of vulvovaginal candidiasis from 1996 to 2017. Data obtained could represent the susceptibility profile of C. albicans strains circulating in Argentina and could be of potential usefulness to monitor and guide therapy, and also suggests the need for greater surveillance programs to detect fluconazole resistance over time.

     

Barriers and Facilitators to the Uptake and Maintenance of Healthy Behaviours by People at Mid-Life: A Rapid Systematic Review

Background

With an ageing population, there is an increasing societal impact of ill health in later life. People who adopt healthy behaviours are more likely to age successfully. To engage people in health promotion initiatives in mid-life, a good understanding is needed of why people do not undertake healthy behaviours or engage in unhealthy ones.

Methods

Searches were conducted to identify systematic reviews and qualitative or longitudinal cohort studies that reported mid-life barriers and facilitators to healthy behaviours. Mid-life ranged from 40 to 64 years, but younger adults in disadvantaged or minority groups were also eligible to reflect potential earlier disease onset. Two reviewers independently conducted reference screening and study inclusion. Included studies were assessed for quality. Barriers and facilitators were identified and synthesised into broader themes to allow comparisons across behavioural risks.

Findings

From 16,426 titles reviewed, 28 qualitative studies, 11 longitudinal cohort studies and 46 systematic reviews were included. Evidence was found relating to uptake and maintenance of physical activity, diet and eating behaviours, smoking, alcohol, eye care, and other health promoting behaviours and grouped into six themes: health and quality of life, sociocultural factors, the physical environment, access, psychological factors, evidence relating to health inequalities. Most of the available evidence was from developed countries. Barriers that recur across different health behaviours include lack of time (due to family, household and occupational responsibilities), access issues (to transport, facilities and resources), financial costs, entrenched attitudes and behaviours, restrictions in the physical environment, low socioeconomic status, lack of knowledge. Facilitators include a focus on enjoyment, health benefits including healthy ageing, social support, clear messages, and integration of behaviours into lifestyle. Specific issues relating to population and culture were identified relating to health inequalities.

Conclusions

The barriers and facilitators identified can inform the design of tailored interventions for people in mid-life.     

Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.     

Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency

Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.     

Proteinaceous inhibitor versus fructose as modulators of Pteris deflexa invertase activity

An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer Mr 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had beta-fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with Km of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.