The cardiomyocyte "redox rheostat": Redox signalling via the AMPK-mTOR axis and regulation of gene and protein expression balancing survival and death.

Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H2O2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H2O2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H2O2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H2O2 levels are cytoprotective. We identified p21Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a "redox rheostat" in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death

Renal screening in children after exposure to low dose melamine in Hong Kong: cross sectional study

Objective To investigate the renal outcomes of children after exposure to low dose melamine in Hong Kong

Discrepancy between Mtb-specific IFN-γ and IgG responses in HIV-positive people with low CD4 counts

Background: Tuberculosis (TB) is a leading infectious cause of death worldwide and treating latent TB infection (LTBI) with TB preventative therapy is a global priority. This study aimed to measure interferon gamma (IFN-γ) release assay (IGRA) positivity (the current reference standard for LTBI diagnosis) and Mtb-specific IgG antibodies in otherwise healthy adults without HIV and those living with HIV (PLWH). Methods: One-hundred and eighteen adults (65 without HIV and 53 antiretroviral-naïve PLWH), from a peri-urban setting in KwaZulu-Natal, South Africa were enrolled. IFN-γ released following stimulation with ESAT-6/CFP-10 peptides and plasma IgG antibodies specific for multiple Mtb antigens were measured using the QuantiFERON-TB Gold Plus (QFT) and customized Luminex assays, respectively. The relationships between QFT status, relative concentrations of anti-Mtb IgG, HIV-status, sex, age and CD4 count were analysed. Findings: Older age, male sex and higher CD4 count were independently associated with QFT positivity (p = 0.045, 0.05 and 0.002 respectively). There was no difference in QFT status between people with and without HIV infection (58% and 65% respectively, p = 0.06), but within CD4 count quartiles, people with HIV had higher QFT positivity than people without HIV (p = 0.008 (2nd quartile), <0.0001 (3rd quartile)). Concentrations of Mtb-specific IFN-γ were lowest, and relative concentrations of Mtb-specific IgGs were highest in PLWH in the lowest CD4 quartile. Interpretation: These results suggest that the QFT assay underestimates LTBI among immunosuppressed people with HIV and Mtb-specific IgG may be a useful alternative biomarker for Mtb infection. Further evaluation of how Mtb-specific antibodies can be leveraged to improve LTBI diagnosis is warranted, particularly in HIV-endemic areas. Fundings: NIH, AHRI, SHIP: SA-MRC and SANTHE

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al

Erratum to: Preclinical activity of gefitinib in non-keratinizing nasopharyngeal carcinoma cell lines and biomarkers of response

published_or_final_versionSpringer Open Choice, 01 Dec 201

Etiology of Childhood Bacteremia and Timely Antibiotics Administration in the Emergency Department

BACKGROUND: Bacteremia is now an uncommon presentation to the children’s emergency department (ED) but is associated with significant morbidity and mortality. Its evolving etiology may affect the ability of clinicians to initiate timely, appropriate antimicrobial therapy. METHODS: A retrospective time series analysis of bacteremia was conducted in the Alder Hey Children’s Hospital ED between 2001 and 2011. Data on significant comorbidities, time to empirical therapy, and antibiotic susceptibility were recorded. RESULTS: A total of 575 clinical episodes were identified, and Streptococcus pneumoniae (n = 109), Neisseria meningitidis (n = 96), and Staphylococcus aureus (n = 89) were commonly isolated. The rate of bacteremia was 1.42 per 1000 ED attendances (95% confidence interval: 1.31–1.53). There was an annual reduction of 10.6% (6.6%–14.5%) in vaccine-preventable infections, and an annual increase of 6.7% (1.2%–12.5%) in Gram-negative infections. The pneumococcal conjugate vaccine was associated with a 49% (32%–74%) reduction in pneumococcal bacteremia. The rate of health care–associated bacteremia increased from 0.17 to 0.43 per 1000 ED attendances (P = .002). Susceptibility to empirical antibiotics was reduced (96.3%–82.6%; P < .001). Health care–associated bacteremia was associated with an increased length of stay of 3.9 days (95% confidence interval: 2.3–5.8). Median time to antibiotics was 184 minutes (interquartile range: 63–331) and 57 (interquartile range: 27–97) minutes longer in Gram-negative bacteremia than in vaccine-preventable bacteremia. CONCLUSIONS: Changes in the etiology of pediatric bacteremia have implications for prompt, appropriate empirical treatment. Increasingly, pediatric bacteremia in the ED is health care associated, which increases length of inpatient stay. Prompt, effective antimicrobial administration requires new tools to improve recognition, in addition to continued etiological surveillance

Facing the threat of influenza pandemic - roles of and implications to general practitioners

The 2009 pandemic of H1N1 influenza, compounded with seasonal influenza, posed a global challenge. Despite the announcement of post-pandemic period on 10 August 2010 by theWHO, H1N1 (2009) virus would continue to circulate as a seasonal virus for some years and national health authorities should remain vigilant due to unpredictable behaviour of the virus. Majority of the world population is living in countries with inadequate resources to purchase vaccines and stockpile antiviral drugs. Basic hygienic measures such as wearing face masks and the hygienic practice of hand washing could reduce the spread of the respiratory viruses. However, the imminent issue is translating these measures into day-to-day practice. The experience from Severe Acute Respiratory Syndrome (SARS) in Hong Kong has shown that general practitioners (GPs) were willing to discharge their duties despite risks of getting infected themselves. SARS event has highlighted the inadequate interface between primary and secondary care and valuable health care resources were thus inappropriately matched to community needs