Influence of mutagenesis and viral load on the sustained low-level replication of an RNA virus

Lethal mutagenesis is an antiviral strategy that aims to extinguish viruses as a consequence of enhanced mutation rates during virus replication. The molecular mechanisms that underlie virus extinction by mutagenic nucleoside analogues are not well understood. When mutagenic agents and antiviral inhibitors are administered sequentially or in combination, interconnected and often conflicting selective constraints can influence the fate of the virus either towards survival through selection of mutagen-escape or inhibitor-escape mutants or towards extinction. Here we report a study involving the mutagenesis of foot-and-mouth disease virus (FMDV) by the nucleoside analogue ribavirin (R) and the effect of R-mediated mutagenesis on the selection of FMDV mutants resistant to the inhibitor of RNA replication, guanidine hydrochloride (GU). The results show that under comparable (and low) viral load, an inhibitory activity by GU could not substitute for an equivalent inhibitory activity by R in driving FMDV to extinction. Both the prior history of R mutagenesis and the viral population size influenced the selection of GU-escape mutants. A sufficiently low viral load allowed continued viral replication without selection of inhibitor-escape mutants, irrespective of the history of mutagenesis. These observations imply that reductions of viral load as a result of a mutagenic treatment may provide an opportunity either for immune-mediated clearing of a virus or for an alternative antiviral intervention, even if extinction is not initially achieved.     

Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay

Using 16S rRNA gene sequencing analysis, we examined the bacterial diversity and the presence of opportunistic bacterial pathogens (i.e., Campylobacter and Helicobacter) in red knot (Calidris canutus; n = 40), ruddy turnstone (Arenaria interpres; n = 35), and semipalmated sandpiper (Calidris pusilla; n = 22) fecal samples collected during a migratory stopover in Delaware Bay. Additionally, we studied the occurrence of Campylobacter spp., enterococci, and waterfowl fecal source markers using quantitative PCR (qPCR) assays. Of 3,889 16S rRNA clone sequences analyzed, the bacterial community was mostly composed of Bacilli (63.5%), Fusobacteria (12.7%), Epsilonproteobacteria (6.5%), and Clostridia (5.8%). When epsilonproteobacterium-specific 23S rRNA gene clone libraries (i.e., 1,414 sequences) were analyzed, the sequences were identified as Campylobacter (82.3%) or Helicobacter (17.7%) spp. Specifically, 38.4%, 10.1%, and 26.0% of clone sequences were identified as C. lari (>99% sequence identity) in ruddy turnstone, red knot, and semipalmated sandpiper clone libraries, respectively. Other pathogenic species of Campylobacter, such as C. jejuni and C. coli, were not detected in excreta of any of the three bird species. Most Helicobacter-like sequences identified were closely related to H. pametensis (>99% sequence identity) and H. anseris (92% sequence identity). qPCR results showed that the occurrence and abundance of Campylobacter spp. was relatively high compared to those of fecal indicator bacteria, such as Enterococcus spp., E. faecalis, and Catellicoccus marimammalium. Overall, the results provide insights into the complexity of the shorebird gut microbial community and suggest that these migratory birds are important reservoirs of pathogenic Campylobacter species.     

C‐terminal β‐strand swapping in a consensus‐derived fibronectin Type III scaffold

The crystal structures of six different fibronectin Type III consensus‐derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C‐terminal β‐strand resulting in the formation of Tencon dimers in which the target‐binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the β‐strand‐swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non‐glycine residue at position 77 and insertions of <4 residues may destabilize the β‐turn in the FG loop promoting β‐strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect β‐bridges in the FG regions that link the swapped dimers. The Tencon β‐swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain‐swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties. Proteins 2014; 82:1359–1369. © 2013 Wiley Periodicals, Inc.     

Has the Prevalence of Migraine Changed over the Last Decade (2003–2012)? A Spanish Population-Based Survey

Introduction

Information on temporal trends can identify groups of people at risk for any particular condition; however information on temporal trends on migraine headache at population levels is scarce. Our aim was to estimate the time trends in the prevalence of migraine from 2003 to 2012 in Spain.

Methods

A population-based national study was conducted. We analyzed data using individualized information taken from national surveys conducted in 2003/4, 2006/7, 2009/10 and 2011/12. A total of 94,158 Spanish adults participated. We considered the presence of self-rated and diagnosed migraine, and we analyzed socio-demographic features, lifestyle habits, self-rated health status, and comorbid diseases using logistic regressions.

Results

The prevalence of migraine increased from 6.54% in 2003 to 9.69% in 2012 with significant time trends (adj. OR 1.65; 95%CI 1.50–1.81). The probability of women of suffering migraine was 3 times higher than for men (adj.OR 3.08; 2.82–3.37). There was a declining trend in migraine prevalence as age increased (adj.OR 0.42; 0.35–0.51). Demographic variables associated with migraine were lower educational level (adj.OR 1.32; 1.13–1.54) and not being an immigrant (adj.OR 1.37; 1.15–1.64). A worse self-reported health status was related to higher prevalence of migraine (adj.OR 2.83; 2.59–3.09). The prevalence of migraine also increased as the number of comorbid conditions increased (adj.OR 2.42; 2.05–2.86).

Conclusion

The prevalence of migraine has increased in the first decade of the 21^st century in Spain. Migraine was associated with being female, mid-age, low educational level, not being an immigrant, worse self-rated health status and presence of comorbid conditions.     

Ecological functioning in grass–shrub Mediterranean ecosystems measured by eddy covariance

Climate change may alter ecosystem functioning, as assessed via the net carbon (C) exchange (NEE) with the atmosphere, composed of the biological processes photosynthesis (GPP) and respiration (R _eco). In addition, in semi-arid Mediterranean ecosystems, a significant fraction of respired CO₂ is stored in the vadose zone and emitted afterwards by subsoil ventilation (VE), contributing also to NEE. Such conditions complicate the prediction of NEE for future change scenarios. To evaluate the possible effects of climate change on annual NEE and its underlying processes (GPP, R _eco and VE) we present, over a climate/altitude range, the annual and interannual variability of NEE, GPP, R _eco and VE in three Mediterranean sites. We found that annual NEE varied from a net source of around 130 gC m^?2 in hot and arid lowlands to a net sink of similar magnitude for alpine meadows (above 2,000 m a.s.l) that are less water stressed. Annual net C fixation increased because of increased GPP during intermittent and several growth periods occurring even during winter, as well as due to decreased VE. In terms of interannual variability, the studied subalpine site behaved as a neutral C sink (from emission of 49 to fixation of 30 gC m^?2 year^?1), with precipitation as the main factor controlling annual GPP and R _eco. Finally, the importance of VE as 0–23 % of annual NEE is highlighted, indicating that this process could shift some Mediterranean ecosystems from annual C sinks to sources.     

Measuring and modelling the radiation balance of a heterogeneous shrubland

The accurate quantification of the energy available for sensible and latent heat transfer from plant canopies is essential for the prediction of impacts of climate on vegetation water use and growth. Unlike agricultural fields and extensive forests of more humid zones, vegetation growing in semi‐arid climates is usually sparse creating a heterogeneous surface of shrubs, annuals and bare soil. Under these conditions many of the assumptions of the basic equations used in microclimatology, which assume a uniform vegetated surface, may be violated. It is proposed here that heterogeneous canopies may require a formulation of their energy balance that includes a measure of the canopy complexity in order to both interpret field measurements and be used in predictive models. This paper explores the need for a more complex formulation of the vegetation energy balance through a series of experiments on a sparse clumped shrubland of Retama sphaerocarpa in the Tabernas Desert, Almería, south‐east Spain. These experiments investigated the importance of the radiative properties of each surface on energy balance of soil, annuals and shrubs individually, and the surface as a whole. The study evaluated the use of the fractional vegetative cover (f) and the radiative characteristics of each surface (reflection coefficients and emissivities) for calculating net radiation partitioning between shrubs and bare soil. Results indicated that partitioning of net radiation between components could be accurately calculated from values of fractional vegetative cover, reflection coefficients and emissivities for both bare soil and plant surfaces. A sensitivity analysis showed the importance of specific radiation properties of each surface. Measurements of horizontal long‐wave fluxes between components showed that the location of a plant with respect to other plants made little difference to its long‐wave energy balance. The results also emphasized the importance of soil water content on the energy balance, through its influence on albedo and soil heat storage. This was particularly true when measurements of soil heat flux were used to measure the available energy for soil under shrubs and bare soil because of strong hysteresis cycles. These cycles were larger in the bare soil than in the substrate under shrubs.     

A phenomenological computational model of the evoked action potential fitted to human cochlear implant responses

There is a growing interest in biomedical engineering in developing procedures that provide accurate simulations of the neural response to electrical stimulus produced by implants. Moreover, recent research focuses on models that take into account individual patient characteristics.We present a phenomenological computational model that is customized with the patient’s data provided by the electrically evoked compound action potential (ECAP) for simulating the neural response to electrical stimulus produced by the electrodes of cochlear implants (CIs). The model links the input currents of the electrodes to the simulated ECAP.Potentials and currents are calculated by solving the quasi-static approximation of the Maxwell equations with the finite element method (FEM). In ECAPs recording, an active electrode generates a current that elicits action potentials in the surrounding auditory nerve fibers (ANFs). The sum of these action potentials is registered by other nearby electrode. Our computational model emulates this phenomenon introducing a set of line current sources replacing the ANFs by a set of virtual neurons (VNs). To fit the ECAP amplitudes we assign a suitable weight to each VN related with the probability of an ANF to be excited. This probability is expressed by a cumulative beta distribution parameterized by two shape parameters that are calculated by means of a differential evolution algorithm (DE). Being the weights function of the current density, any change in the design of the CI affecting the current density produces changes in the weights and, therefore, in the simulated ECAP, which confers to our model a predictive capacity.The results of the validation with ECAP data from two patients are presented, achieving a satisfactory fit of the experimental data with those provided by the proposed computational model.     

Human NK cell-mediated cytotoxicity triggered by CD86 and Gal alpha 1,3-Gal is inhibited in genetically modified porcine cells

Delayed xenograft rejection is a major hurdle that needs to be addressed to prolong graft survival in pig-to-primate xenotransplantation. NK cell activation has been implicated in delayed xenograft rejection. Both Ab-dependent and independent mechanisms are responsible for the high susceptibility of porcine cells to human NK cell-mediated cytotoxicity. Previous reports demonstrated a role of Galalpha1,3-Gal Ag in triggering the Ab-independent responses. We hypothesize that expression of CD80 and/or CD86 on porcine cells may also play a role in NK cell activation as human NK cells express a variant of CD28. Our initial analysis showed that porcine endothelial cells and fibroblasts express CD86, but not CD80. Genetic engineering of these cells to express hCD152-hCD59, a chimeric molecule designed to block CD86 in cis, was accompanied by a reduction in susceptibility to human NK cell-mediated cytotoxicity. The use of a specific anti-porcine CD86-blocking Ab and the NK92 and YTS cell lines further confirmed the involvement of CD86 in triggering NK cell-mediated lysis of porcine cells. Maximal protection was achieved when hCD152-hCD59 was expressed in H transferase-transgenic cells, which show reduced Galalpha1,3-Gal expression. In this work, we describe two mechanisms of human NK cell-mediated rejection of porcine cells and demonstrate that genetically modified cells resist Ab-independent NK cell-mediated cytotoxicity.