Study of cosolvent-induced α-chymotrypsin fibrillogenesis: Does protein surface hydrophobicity trigger early stages of aggregation reaction?

The misfolding of specific proteins is often associated with their assembly into fibrillar aggregates, commonly termed amyloid fibrils. Despite the many efforts expended to characterize amyloid formation in vitro, there is no deep knowledge about the environment (in which aggregation occurs) as well as mechanism of this type of protein aggregation. Alpha-chymotrypsin was recently driven toward amyloid aggregation by the addition of intermediate concentrations of trifluoroethanol. In the present study, approaches such as turbidimetric, thermodynamic, intrinsic fluorescence and quenching studies as well as chemical modification have been successfully used to elucidate the underlying role of hydrophobic interactions (involved in early stages of amyloid formation) in α-chymotrypsin-based experimental system. © 2009 Springer Science+Business Media, LLC

Genome-wide transcriptional profiling of appressorium development by the rice blast fungus Magnaporthe oryzae.

addresses: College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom.notes: PMCID: PMC3276559The rice blast fungus Magnaporthe oryzae is one of the most significant pathogens affecting global food security. To cause rice blast disease the fungus elaborates a specialised infection structure called an appressorium. Here, we report genome wide transcriptional profile analysis of appressorium development using next generation sequencing (NGS). We performed both RNA-Seq and High-Throughput SuperSAGE analysis to compare the utility of these procedures for identifying differential gene expression in M. oryzae. We then analysed global patterns of gene expression during appressorium development. We show evidence for large-scale gene expression changes, highlighting the role of autophagy, lipid metabolism and melanin biosynthesis in appressorium differentiation. We reveal the role of the Pmk1 MAP kinase as a key global regulator of appressorium-associated gene expression. We also provide evidence for differential expression of transporter-encoding gene families and specific high level expression of genes involved in quinate uptake and utilization, consistent with pathogen-mediated perturbation of host metabolism during plant infection. When considered together, these data provide a comprehensive high-resolution analysis of gene expression changes associated with cellular differentiation that will provide a key resource for understanding the biology of rice blast disease

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals toxic effects observed.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes" (NORTE-07-0124-FEDER-000028), Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. Manuela D. Machado gratefully acknowledges the post-doctoral grant from FCT (SFRH/BPD/72816/2010). Vanessa A. Mesquita gratefully acknowledges the grant from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES). The authors also thank to Doctor Rosane Freitas Schwan to offer the yeast strain and to Doctor Helena M.V.M. Soares, from the Faculty of Engineering of Porto University, for the use of analytical facilities (AAS with flame atomization and AAS with electrothermal atomization)

Human immunodeficiency virus seroconversion presenting with acute inflammatory demyelinating polyneuropathy: a case report

<p>Abstract</p> <p>Introduction</p> <p>Acute Human Immunodeficiency Virus infection is associated with a range of neurological conditions. Guillain-Barré syndrome is a rare presentation; acute inflammatory demyelinating polyneuropathy is the commonest form of Guillain-Barré syndrome. Acute inflammatory demyelinating polyneuropathy has occasionally been reported in acute Immunodeficiency Virus infection but little data exists on frequency, management and outcome.</p> <p>Case presentation</p> <p>We describe an episode of Guillain-Barré syndrome presenting as acute inflammatory demyelinating polyneuropathy in a 30-year-old man testing positive for Immunodeficiency Virus, probably during acute seroconversion. Clinical suspicion was confirmed by cerebrospinal fluid analysis and nerve conduction studies. Rapid clinical deterioration prompted intravenous immunoglobulin therapy and early commencement of highly active anti-retroviral therapy. All symptoms resolved within nine weeks.</p> <p>Conclusion</p> <p>Unusual neurological presentations in previously fit patients are an appropriate indication for Immunodeficiency-Virus testing. Highly active anti-retroviral therapy with adequate penetration of the central nervous system should be considered as an early intervention, alongside conventional therapies such as intravenous immunoglobulin.</p

Helicobacter pylori CagA Triggers Expression of the Bactericidal Lectin REG3γ via Gastric STAT3 Activation

Background: Most of what is known about the Helicobacter pylori (H. pylori) cytotoxin, CagA, pertains to a much-vaunted role as a determinant of gastric inflammation and cancer. Little attention has been devoted to potential roles of CagA in the majority of H. pylori infected individuals not showing oncogenic progression, particularly in relation to host tolerance. Regenerating islet-derived (REG)3c encodes a secreted C-type lectin that exerts direct bactericidal activity against Grampositive bacteria in the intestine. Here, we extend this paradigm of lectin-mediated innate immunity, showing that REG3c expression is triggered by CagA in the H. pylori-infected stomach. Methodology/Principal Findings: In human gastric mucosal tissues, REG3c expression was significantly increased in CagApositive, compared to CagA-negative H. pylori infected individuals. Using transfected CagA-inducible gastric MKN28 cells, we recapitulated REG3c induction in vitro, also showing that tyrosine phosphorylated, not unphosphorylated CagA triggers REG3c transcription. In concert with induced REG3c, pro-inflammatory signalling downstream of the gp130 cytokine coreceptor via the signal transducer and activator of transcription (STAT)3 and transcription of two cognate ligands, interleukin(IL)-11 and IL-6, were significantly increased. Exogenous IL-11, but not IL-6, directly stimulated STAT3 activation and REG3c transcription. STAT3 siRNA knockdown or IL-11 receptor blockade respectively abrogated or subdued CagAdependent REG3c mRNA induction, thus demonstrating a requirement for uncompromised signalling via the IL-11/STAT

Presenilin Is the Molecular Target of Acidic γ-Secretase Modulators in Living Cells

The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-β (Aβ) peptides and is a principal therapeutic target in Alzheimer's disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs), which are small molecules that selectively lower generation of the highly amyloidogenic Aβ42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP) and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aβ42 generation with an IC50 of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN), the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex

Gender- and Age-Dependent γ-Secretase Activity in Mouse Brain and Its Implication in Sporadic Alzheimer Disease

Alzheimer disease (AD) is an age-related disorder. Aging and female gender are two important risk factors associated with sporadic AD. However, the mechanism by which aging and gender contribute to the pathogenesis of sporadic AD is unclear. It is well known that genetic mutations in γ-secretase result in rare forms of early onset AD due to the aberrant production of Aβ42 peptides, which are the major constituents of senile plaques. However, the effect of age and gender on γ-secretase has not been fully investigated. Here, using normal wild-type mice, we show mouse brain γ-secretase exhibits gender- and age-dependent activity. Both male and female mice exhibit increased Aβ42∶Aβ40 ratios in aged brain, which mimics the effect of familial mutations of Presenilin-1, Presenlin-2, and the amyloid precursor protein on Aβ production. Additionally, female mice exhibit much higher γ-secretase activity in aged brain compared to male mice. Furthermore, both male and female mice exhibit a steady decline in Notch1 γ-secretase activity with aging. Using a small molecule affinity probe we demonstrate that male mice have less active γ-secretase complexes than female mice, which may account for the gender-associated differences in activity in aged brain. These findings demonstrate that aging can affect γ-secretase activity and specificity, suggesting a role for γ-secretase in sporadic AD. Furthermore, the increased APP γ-secretase activity seen in aged females may contribute to the increased incidence of sporadic AD in women and the aggressive Aβ plaque pathology seen in female mouse models of AD. In addition, deceased Notch γ-secretase activity may also contribute to neurodegeneration. Therefore, this study implicates altered γ-secretase activity and specificity as a possible mechanism of sporadic AD during aging

Understanding the (non-)Use of Societal Wellbeing Indicators in National Policy Development : What Can We Learn from Civil Servants? A UK Case Study

Gross Domestic Product is often used as a proxy for societal well-being in the context of policy development. Its shortcomings in this context are, however, well documented, and numerous alternative indicator sets have been developed. Despite this, there is limited evidence of widespread use of these alternative indicator sets by people working in policy areas relevant to societal wellbeing. Civil servants are an important group of indicator end-users. Better understanding their views concerning measuring societal wellbeing can support wider discussions about what factors determine indicator use and influence in policy decision-making. Taking the UK as a case study, we ask what views exist among civil servants in the UK about measuring societal well-being? To answer this question, we used a bootstrapped Q methodology, interviewing 20 civil servants to elicit their views about measuring societal well-being. Three distinct discourses emerged from our analysis: one that was concerned about the consequences of ignoring natural, social and human capital in decision making; one that emphasised opportunity and autonomy as key determinants of well-being; and one that focused on the technical aspects of measuring societal well-being. Each of these discourses has direct implications for the way that we integrate societal wellbeing into policy making and highlights the potential benefits of including end-users in indicator development and strategy