Characterization of the Zn(II) Binding Properties of the Human Wilms’ Tumor Suppressor Protein C-terminal Zinc Finger Peptide

Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ββα protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ββα folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/ββα zinc finger domain, the C-terminal finger of the Wilms’ tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)–WT1-4 followed by fluorescence spectroscopy yielded the effective pKaeff values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKaeff values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)–WT1-4, KfML value of 7.5 × 1012 M–1, with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least −17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)–WT1-4 to those from the model peptide Zn(II)–GGG–Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent KfML values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc finger proteins and support the conclusion that the cost of protein folding in most zinc finger proteins is ≤+4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, −17.6 kcal/mol

Trajectories of self-rated health in people with diabetes: Associations with functioning in a prospective community sample

© 2013 Schmitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Self-rated health (SRH) is a single-item measure that is one of the most widely used measures of general health in population health research. Relatively little is known about changes and the trajectories of SRH in people with chronic medical conditions. The aims of the present study were to identify and describe longitudinal trajectories of self-rated health (SRH) status in people with diabetes. Methods: A prospective community study was carried out between 2008 and 2011. SRH was assessed at baseline and yearly at follow-ups (n=1288). Analysis was carried out through trajectory modeling. The trajectory groups were subsequently compared at 4 years follow-up with respect to functioning. Results: Four distinct trajectories of SRH were identified: 1) 72.2% of the participants were assigned to a persistently good SRH trajectory; 2) 10.1% were assigned to a persistently poor SRH trajectory; 3) mean SRH scores changed from good to poor for one group (7.3%); while 4) mean SRH scores changed from poor to medium/good for another group (10.4%). Those with a persistently poor perception of health status were at higher risk for poor functioning at 4 years follow-up than those whose SRH scores decreased from good to poor. Conclusions: SRH is an important predictor for poor functioning in diabetes, but the trajectory of SRH seems to be even more important. Health professionals should pay attention to not only SRH per se, but also changes in SRH over time.This work was supported by Operating Grant MOP-84574 from the Canadian Institutes of Health Research (CIHR). GG was supported by a doctoral fellowship from the CIHR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Functional interface micromechanics of 11 en-bloc retrieved cemented femoral hip replacements

Contains fulltext : 88556.pdf (publisher's version ) (Open Access)BACKGROUND AND PURPOSE: Despite the longstanding use of micromotion as a measure of implant stability, direct measurement of the micromechanics of implant/bone interfaces from en bloc human retrievals has not been performed. The purpose of this study was to determine the stem-cement and cement-bone micromechanics of functionally loaded, en-bloc retrieved, cemented femoral hip components. METHODS: 11 fresh frozen proximal femurs with cemented implants were retrieved at autopsy. Specimens were sectioned transversely into 10-mm slabs and fixed to a loading device where functional torsional loads were applied to the stem. A digital image correlation technique was used to document micromotions at stem-cement and cement-bone interfaces during loading. RESULTS: There was a wide range of responses with stem-cement micromotions ranging from 0.0006 mm to 0.83 mm (mean 0.17 mm, SD 0.29) and cement-bone micromotions ranging from 0.0022 mm to 0.73 mm (mean 0.092 mm, SD 0.22). There was a strong (linear-log) inverse correlation between apposition fraction and micromotion at the stem-cement interface (r(2) = 0.71, p < 0.001). There was a strong inverse log-log correlation between apposition fraction at the cement-bone interface and micromotion (r(2) = 0.85, p < 0.001). Components that were radiographically well-fixed had a relatively narrow range of micromotions at the stem-cement (0.0006-0.057 mm) and cement-bone (0.0022-0.029 mm) interfaces. INTERPRETATION: Minimizing gaps at the stem-cement interface and encouraging bony apposition at the cement-bone interface would be clinically desirable. The cement-bone interface does not act as a bonded interface in actual use, even in radiographically well-fixed components. Rather, the interface is quite compliant, with sliding and opening motions between the cement and bone surfaces.1 juni 201

IFITM Proteins Restrict Antibody-Dependent Enhancement of Dengue Virus Infection

Interferon-inducible transmembrane (IFITM) proteins restrict the entry processes of several pathogenic viruses, including the flaviviruses West Nile virus and dengue virus (DENV). DENV infects cells directly or via antibody-dependent enhancement (ADE) in Fc-receptor-bearing cells, a process thought to contribute to severe disease in a secondary infection. Here we investigated whether ADE-mediated DENV infection bypasses IFITM-mediated restriction or whether IFITM proteins can be protective in a secondary infection. We observed that IFITM proteins restricted ADE-mediated and direct infection with comparable efficiencies in a myelogenous leukemia cell line. Our data suggest that IFITM proteins can contribute to control of secondary DENV infections

Health differentials in the older population of England: An empirical comparison of the materialist, lifestyle and psychosocial hypotheses

BACKGROUND: In developed countries with old age structures most deaths occur at older ages and older people account for the majority of those in poor health, which suggests a particular need to investigate health inequalities in the older population. METHODS: We empirically compared the materialist, psychosocial and lifestyle/behavioural theoretical mechanisms of explanation for socio-economic variation in health using data from two waves of the English Longitudinal Study of Ageing (ELSA), a nationally representative multi-purpose sample of the population aged 50 and over living in England. Three dimensions of health were examined: somatic health, depression and well-being. RESULTS: The materialist and lifestyle/behavioural paths had the most prominent mediating role in the association between socio-economic position and health in the older population, whereas the psychosocial pathway was less influential and exerted most of its influence on depression and well-being, with part of its effect being due to the availability of material resources. CONCLUSIONS: From a policy perspective there is therefore an indication that population interventions to reduce health differentials and thus improve the overall health of the older population should focus on material circumstances and population based interventions to promote healthy lifestyles

Impact of plants on the diversity and activity of methylotrophs in soil

Background Methanol is the second most abundant volatile organic compound in the atmosphere, with the majority produced as a metabolic by-product during plant growth. There is a large disparity between the estimated amount of methanol produced by plants and the amount which escapes to the atmosphere. This may be due to utilisation of methanol by plant-associated methanol-consuming bacteria (methylotrophs). The use of molecular probes has previously been effective in characterising the diversity of methylotrophs within the environment. Here, we developed and applied molecular probes in combination with stable isotope probing to identify the diversity, abundance and activity of methylotrophs in bulk and in plant-associated soils. Results Application of probes for methanol dehydrogenase genes (mxaF, xoxF, mdh2) in bulk and plant-associated soils revealed high levels of diversity of methylotrophic bacteria within the bulk soil, including Hyphomicrobium, Methylobacterium and members of the Comamonadaceae. The community of methylotrophic bacteria captured by this sequencing approach changed following plant growth. This shift in methylotrophic diversity was corroborated by identification of the active methylotrophs present in the soils by DNA stable isotope probing using 13C-labelled methanol. Sequencing of the 16S rRNA genes and construction of metagenomes from the 13C-labelled DNA revealed members of the Methylophilaceae as highly abundant and active in all soils examined. There was greater diversity of active members of the Methylophilaceae and Comamonadaceae and of the genus Methylobacterium in plant-associated soils compared to the bulk soil. Incubating growing pea plants in a 13CO2 atmosphere revealed that several genera of methylotrophs, as well as heterotrophic genera within the Actinomycetales, assimilated plant exudates in the pea rhizosphere. Conclusion In this study, we show that plant growth has a major impact on both the diversity and the activity of methanol-utilising methylotrophs in the soil environment, and thus, the study contributes significantly to efforts to balance the terrestrial methanol and carbon cycle

Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model.

Zika virus is a mosquito-borne flavivirus closely related to dengue virus that can cause severe disease in humans, including microcephaly in newborns and Guillain-Barré syndrome in adults. Specific treatments and vaccines for Zika virus are not currently available. Here, we isolate and characterize four monoclonal antibodies (mAbs) from an infected patient that target the non-structural protein NS1. We show that while these antibodies are non-neutralizing, NS1-specific mAbs can engage FcγR without inducing antibody dependent enhancement (ADE) of infection in vitro. Moreover, we demonstrate that mAb AA12 has protective efficacy against lethal challenges of African and Asian lineage strains of Zika virus in Stat2-/- mice. Protection is Fc-dependent, as a mutated antibody unable to activate known Fc effector functions or complement is not protective in vivo. This study highlights the importance of the ZIKV NS1 protein as a potential vaccine antigen

Management practices for control of ragwort species

The ragwort species common or tansy ragwort (Jacobaea vulgaris, formerly Senecio jacobaea), marsh ragwort (S. aquaticus), Oxford ragwort (S. squalidus) and hoary ragwort (S. erucifolius) are native in Europe, but invaded North America, Australia and New Zealand as weeds. The abundance of ragwort species is increasing in west-and central Europe. Ragwort species contain different groups of secondary plant compounds defending them against generalist herbivores, contributing to their success as weeds. They are mainly known for containing pyrrolizidine alkaloids, which are toxic to grazing cattle and other livestock causing considerable losses to agricultural revenue. Consequently, control of ragwort is obligatory by law in the UK, Ireland and Australia. Commonly used management practices to control ragwort include mechanical removal, grazing, pasture management, biological control and chemical control. In this review the biology of ragwort species is shortly described and the different management practices are discussed

Novel facultative Methylocella strains are active methane consumers at terrestrial natural gas seeps

Natural gas seeps contribute to global climate change by releasing substantial amounts of the potent greenhouse gas methane and other climate-active gases including ethane and propane to the atmosphere. However, methanotrophs, bacteria capable of utilising methane as the sole source of carbon and energy, play a significant role in reducing the emissions of methane from many environments. Methylocella-like facultative methanotrophs are a unique group of bacteria that grow on other components of natural gas (i.e. ethane and propane) in addition to methane but a little is known about the distribution and activity of Methylocella in the environment. The purposes of this study were to identify bacteria involved in cycling methane emitted from natural gas seeps and, most importantly, to investigate if Methylocella-like facultative methanotrophs were active utilisers of natural gas at seep sites

After the epidemic: Zika virus projections for Latin America and the Caribbean

Background: Zika is one of the most challenging emergent vector-borne diseases, yet its future public health impact remains unclear. Zika was of little public health concern until recent reports of its association with congenital syndromes. By 3 August 2017 ~217,000 Zika cases and ~3,400 cases of associated congenital syndrome were reported in Latin America and the Caribbean. Some modelling exercises suggest that Zika virus infection could become endemic in agreement with recent declarations from the The World Health Organisation. Methodology/Principal findings: We produced high-resolution spatially-explicit projections of Zika cases, associated congenital syndromes and monetary costs for Latin America and the Caribbean now that the epidemic phase of the disease appears to be over. In contrast to previous studies which have adopted a modelling approach to map Zika potential, we project case numbers using a statistical approach based upon reported dengue case data as a Zika surrogate. Our results indicate that ~12.3 (0.7–162.3) million Zika cases could be expected across Latin America and the Caribbean every year, leading to ~64.4 (0.2–5159.3) thousand cases of Guillain-Barré syndrome and ~4.7 (0.0–116.3) thousand cases of microcephaly. The economic burden of these neurological sequelae are estimated to be USD ~2.3 (USD 0–159.3) billion per annum. Conclusions/Significance: Zika is likely to have significant public health consequences across Latin America and the Caribbean in years to come. Our projections inform regional and federal health authorities, offering an opportunity to adapt to this public health challenge