Prevalence of airflow limitation in outpatients with cardiovascular diseases in Japan.

Background and objectives: Cardiovascular disease (CVD) and chronic obstructive pulmonary disease (COPD) commonly coexist and share common risk factors. The prevalence of COPD in outpatients with a smoking history and CVD in Japan is unknown. The aim of this study was to determine the proportion of Japanese patients with a smoking history being treated for CVD who have concurrent airflow limitation compatible with COPD. A secondary objective was to test whether the usage of lung function tests performed in the clinic influenced the diagnosis rate of COPD in the patients identified with airflow limitation. Methods: In a multicenter observational prospective study conducted at 17 centers across Japan, the prevalence of airflow limitation compatible with COPD (defined as forced expiratory volume (FEV)1/FEV6 <0.73, by handheld spirometry) was investigated in cardiac outpatients ≥40 years old with a smoking history who routinely visited the clinic for their CVD. Each patient completed the COPD Assessment Test prior to spirometry testing. Results: Data were available for 995 patients with a mean age of 66.6±10.0 years, of whom 95.5% were male. The prevalence of airflow limitation compatible with COPD was 27.0% (n=269), and 87.7% of those patients (n=236) did not have a prior diagnosis of COPD. The prevalence of previously diagnosed airflow limitation was higher in sites with higher usage of lung function testing (14.0%, 15.2% respectively) compared against sites where it is performed seldom (11.1%), but was still low. Conclusion: The prevalence of airflow limitation in this study indicates that a quarter of outpatients with CVD have COPD, almost all of whom are undiagnosed. This suggests that it is important to look routinely for COPD in CVD outpatients

Mutational analysis of Sse1 (Hsp110) suggests an integral role for this chaperone in yeast prion propagation in vivo.

The yeast Hsp110 chaperone Sse1 is a conserved protein that is a noncanonical member of the Hsp70 protein superfamily. Sse1 influences the cellular response to heat stress and has also been implicated in playing a role in the propagation of prions in yeast. Sse1 can seemingly exert its effects in vivo through direct or indirect actions by influencing the nucleotide exchange activity of canonical cytosolic Hsp70s. Using a genetic screen based on the inability to propagate the yeast [PSI(+)] prion, we have identified 13 new Sse1 mutants that are predicted to alter chaperone function through a variety of different mechanisms. Not only are these new Sse1 mutants altered in the ability to propagate and cure yeast prions but also to varying degrees in the ability to grow at elevated temperatures. The expression levels of chaperone proteins known to influence yeast prion propagation are unaltered in the Sse1 mutants, suggesting that the observed phenotypic effects are caused by direct functional alterations in these mutants. Mapping the location of the mutants onto the Sse1 crystal structure suggests that more than one functional alteration in Sse1 may result in changes in prion propagation and ability to function at elevated temperatures. All Sse1 mutants isolated provide essential functions in the cell under normal growth conditions, further demonstrating that essential chaperone functions in vivo can to some degree at least be detached from those related to propagation of prions. Our results suggest that Sse1 can influence prion propagation through a variety of different mechanisms

Global transcript and phenotypic analysis of yeast cells expressing Ssa1, Ssa2, Ssa3 or Ssa4 as sole source of cytosolic Hsp70-Ssa chaperone activity.

BACKGROUND: Cytosolic Hsp70 is a ubiquitous molecular chaperone that is involved in responding to a variety of cellular stresses. A major function of Hsp70 is to prevent the aggregation of denatured proteins by binding to exposed hydrophobic regions and preventing the accumulation of amorphous aggregates. To gain further insight into the functional redundancy and specialisation of the highly homologous yeast Hsp70-Ssa family we expressed each of the individual Ssa proteins as the sole source of Hsp70 in the cell and assessed phenotypic differences in prion propagation and stress resistance. Additionally we also analysed the global gene expression patterns in yeast strains expressing individual Ssa proteins, using microarray and RT-qPCR analysis. RESULTS: We confirm and extend previous studies demonstrating that cells expressing different Hsp70-Ssa isoforms vary in their ability to propagate the yeast [PSI+] prion, with Ssa3 being the most proficient. Of the four Ssa family members the heat inducible isoforms are more proficient in acquiring thermotolerance and we show a greater requirement than was previously thought, for cellular processes in addition to the traditional Hsp104 protein disaggregase machinery, in acquiring such thermotolerance. Cells expressing different Hsp70-Ssa isoforms also display differences in phenotypic response to exposure to cell wall damaging and oxidative stress agents, again with the heat inducible isoforms providing better protection than constitutive isoforms. We assessed global transcriptome profiles for cells expressing individual Hsp70-Ssa isoforms as the sole source of cytosolic Hsp70, and identified a significant difference in cellular gene expression between these strains. Differences in gene expression profiles provide a rationale for some phenotypic differences we observed in this study. We also demonstrate a high degree of correlation between microarray data and RT-qPCR analysis for a selection of genes. CONCLUSIONS: The Hsp70-Ssa family provide both redundant and variant-specific functions within the yeast cell. Yeast cells expressing individual members of the Hsp70-Ssa family as the sole source of Ssa protein display differences in global gene expression profiles. These changes in global gene expression may contribute significantly to the phenotypic differences observed between the Hsp70-Ssa family members

Towards understanding the gliotoxin detoxification mechanism: in vivo thiomethylation protects yeast from gliotoxin cytotoxicity

Gliotoxin (GT) is a mycotoxin produced by some species of ascomycete fungi including the opportunistic human pathogen Aspergillus fumigatus . In order to produce GT the host organism needs to have evolved a selfprotection mechanism. GT contains a redox-cycling disulfide bridge that is important in mediating toxicity. Recently is has been demonstrated that A. fumigatus possesses a novel thiomethyltransferase protein called GtmA that has the ability to thiomethylate GT in vivo , which aids the organism in regulating GT biosynthesis. It has been suggested that thiomethylation of GT and similar sulfur-containing toxins may play a role in providing self-protection in host organisms. In this work we have engineered Saccharomyces cerevisiae , a GT-naïve organism, to express A. fumigatus GtmA. We demonstrate that GtmA can readily thiomethylate GT in yeast, which results in protection of the organism from exogenous GT. Our work has implications for understanding the evolution of GT self-protection mechanisms in organisms that are GT producers and non-producers

Structural, mechanistic and functional insight into gliotoxin bis-thiomethylation in Aspergillus fumigatus.

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus Self-resistance against gliotoxin is effected by the gliotoxin oxidase GliT, and attenuation of gliotoxin biosynthesis is catalysed by gliotoxin S-methyltransferase GtmA. Here we describe the X-ray crystal structures of GtmA-apo (1.66 Å), GtmA complexed to S-adenosylhomocysteine (1.33 Å) and GtmA complexed to S-adenosylmethionine (2.28 Å), providing mechanistic insights into this important biotransformation. We further reveal that simultaneous elimination of the ability of A. fumigatus to dissipate highly reactive dithiol gliotoxin, via deletion of GliT and GtmA, results in the most significant hypersensitivity to exogenous gliotoxin observed to date. Indeed, quantitative proteomic analysis of ΔgliT::ΔgtmA reveals an uncontrolled over-activation of the gli-cluster upon gliotoxin exposure. The data presented herein reveal, for the first time, the extreme risk associated with intracellular dithiol gliotoxin biosynthesis-in the absence of an efficient dismutation capacity. Significantly, a previously concealed protective role for GtmA and functionality of ETP bis-thiomethylation as an ancestral protection strategy against dithiol compounds is now evident

RNA-seq reveals the pan-transcriptomic impact of attenuating the gliotoxin self-protection mechanism in Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus produces a number of secondary metabolites, one of which, gliotoxin, has been shown to exhibit anti-fungal activity. Thus, A. fumigatus must be able to protect itself against gliotoxin. Indeed one of the genes in the gliotoxin biosynthetic gene cluster in A. fumigatus, gliT, is required for self-protection against the toxin- however the global self-protection mechanism deployed is unclear. RNA-seq was employed to identify genes differentially regulated upon exposure to gliotoxin in A. fumigatus wild-type and A. fumigatus ∆gliT, a strain that is hypersensitive to gliotoxin. RESULTS: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated. Expression of 164 genes was differentially regulated (log2 fold change of 1.5) in A. fumigatus wild-type when exposed to gliotoxin, consisting of 101 genes with up-regulated expression and 63 genes with down-regulated expression. Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin. These consisted of 508 genes with up-regulated expression, and 1192 genes with down-regulated expression. Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism. Specifically, genes involved in gliotoxin biosynthesis, helvolic acid biosynthesis, siderophore-iron transport genes and also nitrogen metabolism genes and ribosome biogenesis genes underwent altered expression. It was confirmed that gliotoxin biosynthesis is induced upon exposure to exogenous gliotoxin, production of unrelated secondary metabolites is attenuated in A. fumigatus ∆gliT, while quantitative proteomic analysis confirmed disrupted translation in A. fumigatus ∆gliT challenged with exogenous gliotoxin. CONCLUSIONS: This study presents the first global investigation of the transcriptional response to exogenous gliotoxin in A. fumigatus wild-type and the hyper-sensitive strain, ∆gliT. Our data highlight the global and extensive affects of exogenous gliotoxin on a sensitive strain devoid of a self-protection mechanism and infer that GliT functionality is required for the optimal biosynthesis of selected secondary metabolites in A. fumigatus

A pilot course for training-in-context in statistics and research methods: Radiation oncology

Objectives. Inadequate training of investigators in statistics and research methods in Africa contributes to having limited local evidence and infrastructure to support multi-centre international trials. Methods of teaching junior oncology professionals tend not to emphasise research discovery, or the roles of emotional engagement and social networking in facilitating effective and efficient learning. We developed a strategy for teaching research methods and statistics in-context, centred on a shared international and practical research project. Design. An African research network (AFRES) was created and members conducted a pilot clinical registry study to acquire real-time data over a 4-month period in 2011. Following study closure, a proto-course consisting of 7 modules, each orientated to a practical topic in study development, implementation and reporting was administered over 18 weeks to all eight AFRES members. A survey of participants was conducted to evaluate the impact of this training strategy. Results. This strictly voluntary project had 5 of 8 AFRES members engaged in the process. Within one year, we generated and submitted two manuscripts and two news items for publication. Participants reported an increased understanding of the principles of evidence-based practice, research methods and interest in pursuing future research initiatives.Conclusion. A novel strategy to build international research infrastructure in Africa, grounded in a practical and relevant project, and which is collaborative and engaging, appears to be efficient and effective

The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation.

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI +] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes

Systems impact of zinc chelation by the epipolythiodioxopiperazine dithiol gliotoxin in Aspergillus fumigatus: a new direction in natural product functionality.

The non-ribosomal peptide gliotoxin, which autoinduces its own biosynthesis, has potent anti-fungal activity, especially in the combined absence of the gliotoxin oxidoreductase GliT and bis-thiomethyltransferase GtmA. Dithiol gliotoxin (DTG) is a substrate for both of these enzymes. Herein we demonstrate that DTG chelates Zn2+ (m/z 424.94), rapidly chelates Zn2+ from Zn(4-(2-pyridylazo)-resorcinol) (Zn(PAR)2) and also inhibits a Zn2+-dependent alkaline phosphatase (AP). Zn2+ addition rescues AP function following DTG-associated inhibition, and pre-incubation of DTG with Zn2+ completely protects AP activity. Zn2+ (1-50 μM) also significantly relieves the potent gliotoxin-mediated inhibition of Aspergillus fumigatus ΔgliT::ΔgtmA (p < 0.05), which infers in vivo dithiol gliotoxin-mediated sequestration of free Zn2+ or chelation from intracellular metalloenzymes as inhibitory mechanisms. Quantitative proteomic analysis revealed that excess Zn2+ alters the effect of gliotoxin on A. fumigatus ΔgliT, with differential abundance of secondary metabolism-associated proteins in the combinatorial condition. GtmA abundance increased 18.8 fold upon co-addition of gliotoxin and Zn2+ compared to gliotoxin alone, possibly to compensate for disruption to GtmA activity, as seen in in vitro assays. Furthermore, DTG effected significant in vitro aggregation of a number of protein classes, including Zn2+-dependent enzymes, while proteins were protected from aggregation by pre-incubating DTG with Zn2+. We conclude that DTG can act in vivo as a Zn2+ chelator, which can significantly impede A. fumigatus growth in the absence of GliT and GtmA

Investigating hyper-vigilance for social threat of lonely children

The hypothesis that lonely children show hypervigilance for social threat was examined in a series of three studies that employed different methods including advanced eye-tracking technology. Hypervigilance for social threat was operationalized as hostility to ambiguously motivated social exclusion in a variation of the hostile attribution paradigm (Study 1), scores on the Children’s Rejection-Sensitivity Questionnaire (Study 2), and visual attention to socially rejecting stimuli (Study 3). The participants were 185 children (11 years-7 months to 12 years-6 months), 248 children (9 years-4 months to 11 years-8 months) and 140 children (8 years-10 months to 12 years-10 months) in the three studies, respectively. Regression analyses showed that, with depressive symptoms covaried, there were quadratic relations between loneliness and these different measures of hypervigilance to social threat. As hypothesized, only children in the upper range of loneliness demonstrated elevated hostility to ambiguously motivated social exclusion, higher scores on the rejection sensitivity questionnaire, and disengagement difficulties when viewing socially rejecting stimuli. We found that very lonely children are hypersensitive to social threat