Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.     

Genotype‐environment associations support a mosaic hybrid zone between two tidal marsh birds

Local environmental features can shape hybrid zone dynamics when hybrids are bounded by ecotones or when patchily distributed habitat types lead to a corresponding mosaic of genotypes. We investigated the role of marsh‐level characteristics in shaping a hybrid zone between two recently diverged avian taxa – Saltmarsh (Ammodramus caudacutus) and Nelson's (A. nelsoni) sparrows. These species occupy different niches where allopatric, with caudacutus restricted to coastal marshes and nelsoni found in a broader array of wetland and grassland habitats and co‐occur in tidal marshes in sympatry. We determined the influence of habitat types on the distribution of pure and hybrid sparrows and assessed the degree of overlap in the ecological niche of each taxon. To do this, we sampled and genotyped 305 sparrows from 34 marshes across the hybrid zone and from adjacent regions. We used linear regression to test for associations between marsh characteristics and the distribution of pure and admixed sparrows. We found a positive correlation between genotype and environmental variables with a patchy distribution of genotypes and habitats across the hybrid zone. Ecological niche models suggest that the hybrid niche was more similar to that of A. nelsoni and habitat suitability was influenced strongly by distance from coastline. Our results support a mosaic model of hybrid zone maintenance, suggesting a role for local environmental features in shaping the distribution and frequency of pure species and hybrids across space.     

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.     

Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression

Cholangiocarcinoma (CCA) has a poor prognosis due to widespread intrahepatic spread. Aspartate β-hydroxylase (ASPH) is a transmembrane protein and catalyzes the hydroxylation of aspartyl and asparaginyl residues in calcium binding epidermal growth factor (cbEGF)-like domains of various proteins, including Notch receptors and ligands. ASPH is highly overexpressed (>95%) in human CCA tumors. We explored the molecular mechanisms by which ASPH mediated the CCA malignant phenotype and evaluated the potential of ASPH as a therapeutic target for CCA. The importance of expression and enzymatic activity of ASPH for CCA growth and progression was examined using shRNA “knockdown” and a mutant construct that reduced its catalytic activity. Second generation small molecule inhibitors (SMIs) of β-hydroxylase activity were developed and used to target ASPH in vitro and in vivo. Subcutaneous and intrahepatic xenograft rodent models were employed to determine anti-tumor effects on CCA growth and development. It was found that the enzymatic activity of ASPH was critical for mediating CCA progression, as well as inhibiting apoptosis. Mechanistically, ASPH overexpression promoted Notch activation and modulated CCA progression through a Notch1-dependent cyclin D1 pathway. Targeting ASPH with shRNAs or a SMI significantly suppressed CCA growth in vivo.     

Transcriptional profiling of epigenetic regulators in somatic embryos during temperature induced formation of an epigenetic memory in Norway spruce

Planta - A significant number of epigenetic regulators were differentially expressed during embryogenesis at different epitype-inducing conditions. Our results support that methylation of DNA and...     

Phosphodiesterase 4 inhibitors and drugs of abuse: current knowledge and therapeutic opportunities

Background

Long-term exposure to drugs of abuse causes an upregulation of the cAMP-signaling pathway in the nucleus accumbens and other forebrain regions, this common neuroadaptation is thought to underlie aspects of drug tolerance and dependence. Phosphodiesterase 4 (PDE4) is an enzyme that the selective hydrolyzes intracellular cAMP. It is expressed in several brain regions that regulate the reinforcing effects of drugs of abuse.

Objective

Here, we review the current knowledge about central nervous system (CNS) distribution of PDE4 isoforms and the effects of systemic and brain-region specific inhibition of PDE4 on behavioral models of drug addiction.

Methods

A systematic literature search was performed using the Pubmed.

Results

Using behavioral sensitization, conditioned place preference and drug self-administration as behavioral models, a large number of studies have shown that local or systemic administration of PDE4 inhibitors reduce drug intake and/or drug seeking for psychostimulants, alcohol, and opioids in rats or mice.

Conclusions

Preclinical studies suggest that PDE4 could be a therapeutic target for several classes of substance use disorder. We conclude by identifying opportunities for the development of subtype-selective PDE4 inhibitors that may reduce addiction liability and minimize the side effects that limit the clinical potential of non-selective PDE4 inhibitors. Several PDE4 inhibitors have been clinically approved for other diseases. There is a promising possibility to repurpose these PDE4 inhibitors for the treatment of drug addiction as they are safe and well-tolerated in patients.

     

A prospective observational cohort study in primary care practices to identify factors associated with treatment failure in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> skin and soft tissue infections

Background

The incidence of outpatient visits for skin and soft tissue infections (SSTIs) has substantially increased over the last decade. The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has made the management of S. aureus SSTIs complex and challenging. The objective of this study was to identify risk factors contributing to treatment failures associated with community-associated S. aureus skin and soft tissue infections SSTIs.

Methods

This was a prospective, observational study among 14 primary care clinics within the South Texas Ambulatory Research Network. The primary outcome was treatment failure within 90 days of the initial visit. Univariate associations between the explanatory variables and treatment failure were examined. A generalized linear mixed-effect model was developed to identify independent risk factors associated with treatment failure.

Results

Overall, 21% (22/106) patients with S. aureus SSTIs experienced treatment failure. The occurrence of treatment failure was similar among patients with methicillin-resistant S. aureus and those with methicillin-susceptible S. aureus SSTIs (19 vs. 24%; p = 0.70). Independent predictors of treatment failure among cases with S. aureus SSTIs was a duration of infection of ≥7 days prior to initial visit [aOR, 6.02 (95% CI 1.74–19.61)] and a lesion diameter size ≥5 cm [5.25 (1.58–17.20)].

Conclusions

Predictors for treatment failure included a duration of infection for ≥7 days prior to the initial visit and a wound diameter of ≥5 cm. A heightened awareness of these risk factors could help direct targeted interventions in high-risk populations.

     

Satellite tracking of gulls and genomic characterization of faecal bacteria reveals environmentally mediated acquisition and dispersal of antimicrobial‐resistant Escherichia coli on the Kenai Peninsula,Alaska

Gulls (Larus spp.) have frequently been reported to carry Escherichia coli exhibiting antimicrobial resistance (AMR E. coli); however, the pathways governing the acquisition and dispersal of such bacteria are not well described. We equipped 17 landfill‐foraging gulls with satellite transmitters and collected gull faecal samples longitudinally from four locations on the Kenai Peninsula, Alaska to assess: (a) gull attendance and transitions between sites, (b) spatiotemporal prevalence of faecally shed AMR E. coli, and (c) genomic relatedness of AMR E. coli isolates among sites. We also sampled Pacific salmon (Oncorhynchus spp.) harvested as part of personal‐use dipnet fisheries at two sites to assess potential contamination with AMR E. coli. Among our study sites, marked gulls most commonly occupied the lower Kenai River (61% of site locations) followed by the Soldotna landfill (11%), lower Kasilof River (5%) and upper Kenai River (<1%). Gulls primarily moved between the Soldotna landfill and the lower Kenai River (94% of transitions among sites), which were also the two locations with the highest prevalence of AMR E. coli. There was relatively high spatial and temporal variability in AMR E. coli prevalence in gull faeces and there was no evidence of contamination on salmon harvested in personal‐use fisheries. We identified E. coli sequence types and AMR genes of clinical importance, with some isolates possessing genes associated with resistance to as many as eight antibiotic classes. Our findings suggest that gulls acquire AMR E. coli at habitats with anthropogenic inputs and subsequent movements may represent pathways through which AMR is dispersed.