ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y₁ receptor agonist, induced a large signal while UTPyS (P2Y₂ agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y₁. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.     

Correction: Overcoming Stagnation in the Levels and Distribution of Child Mortality: The Case of the Philippines

Background

Health-related within-country inequalities continue to be a matter of great interest and concern to both policy makers and researchers. This study aims to assess the level and the distribution of child mortality outcomes in the Philippines across geographical and socioeconomic indicators.

Methodology

Data on 159,130 children ever borne were analysed from five waves of the Philippine Demographic and Health Survey. Direct estimation was used to construct under-five and neonatal mortality rates for the period 1980–2013. Rate differences and ratios, and where possible, slope and relative indices of inequality were calculated to measure disparities on absolute and relative scales. Stratification was undertaken by levels of rural/urban location, island groups and household wealth.

Findings

National under-five and neonatal mortality rates have shown considerable albeit differential reductions since 1980. Recently released data suggests that neonatal mortality has declined following a period of stagnation. Declines in under-five mortality have been accompanied by decreases in wealth and geography-related absolute inequalities. However, relative inequalities for the same markers have remained stable over time. For neonates, mixed evidence suggests that absolute and relative inequalities have remained stable or may have risen.

Conclusion

In addition to continued reductions in under-five mortality, new data suggests that the Philippines have achieved success in addressing the commonly observed stagnated trend in neonatal mortality. This success has been driven by economic improvement since 2006 as well as efforts to implement a nationwide universal health care campaign. Yet, such patterns, nonetheless, accorded with persistent inequalities, particularly on a relative scale. A continued focus on addressing universal coverage, the influence of decentralisation and armed conflict, and issues along the continuum of care is advocated.     

Selectivity is species-dependent: Characterization of standard agonists and antagonists at human,rat, and mouse adenosine receptors

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A₁, A_2A, A_2B, and A₃, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A₁AR of rat and mouse), CGS-21680 (for A_2A AR of rat), and Cl-IB-MECA (for A₃AR of all three species). The functionally selective partial A_2B agonist BAY60-6583 was found to additionally bind to A₁ and A₃AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A₁), preladenant (A_2A), and PSB-603 (A_2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A₃AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9460-9) contains supplementary material, which is available to authorized users.     

Serum Magnesium and Sudden Death in European Hemodialysis Patients

Despite suggestions that higher serum magnesium (Mg) levels are associated with improved outcome, the association with mortality in European hemodialysis (HD) patients has only scarcely been investigated. Furthermore, data on the association between serum Mg and sudden death in this patient group is limited. Therefore, we evaluated Mg in a post-hoc analysis using pooled data from the CONvective TRAnsport STudy (CONTRAST, NCT00205556), a randomized controlled trial (RCT) evaluating the survival risk in dialysis patients on hemodiafiltration (HDF) compared to HD with a mean follow-up of 3.1 years. Serum Mg was measured at baseline and 6, 12, 24 and 36 months thereafter. Cox proportional hazards models, adjusted for confounders using inverse probability weighting, were used to estimate hazard ratios (HRs) of baseline serum Mg on all-cause mortality, cardiovascular mortality, non-cardiovascular mortality and sudden death. A generalized linear mixed model was used to investigate Mg levels over time. Out of 714 randomized patients, a representative subset of 365 (51%) were analyzed in the present study. For every increase in baseline serum Mg of 0.1 mmol/L, the HR for all-cause mortality was 0.85 (95% CI 0.77–94), the HR for cardiovascular mortality 0.73 (95% CI 0.62–0.85) and for sudden death 0.76 (95% CI 0.62–0.93). These findings did not alter after extensive correction for potential confounders, including treatment modality. Importantly, no interaction was found between serum phosphate and serum Mg. Baseline serum Mg was not related to non-cardiovascular mortality. Mg decreased slightly but statistically significant over time (Δ -0.011 mmol/L/year, 95% CI -0.017 to -0.009, p = 0.03). In short, serum Mg has a strong, independent association with all-cause mortality, cardiovascular mortality and sudden death in European HD patients. Serum Mg levels decrease slightly over time.     

Dissonance-Based Eating Disorder Prevention Program Reduces Reward Region Response to Thin Models; How Actions Shape Valuation

Research supports the effectiveness of a dissonance-based eating disorder prevention program wherein high-risk young women with body dissatisfaction critique the thin ideal, which reduces pursuit of this ideal, and the theory that dissonance induction contributes to these effects. Based on evidence that dissonance produces attitudinal change by altering neural representation of valuation, we tested whether completing the Body Project would reduce response of brain regions implicated in reward valuation to thin models. Young women with body dissatisfaction were randomized to this intervention or an educational control condition, completing assessments and fMRI scans while viewing images of thin versus average-weight female models at pre and post. Whole brain analyses indicated that, compared to controls, Body Project participants showed greater reductions in caudate response to images of thin versus average-weight models, though participants in the two conditions showed pretest differences in responsivity of other brain regions that might have contributed to this effect. Greater pre-post reductions in caudate and putamen response to thin models correlated with greater reductions in body dissatisfaction. The finding that the Body Project reduces caudate response to thin models provides novel preliminary evidence that this intervention reduces valuation of media images thought to contribute to body dissatisfaction and eating disorders, providing support for the intervention theory by documenting that this intervention alters an objective biological outcome.     

Genome-Based Identification of Active Prophage Regions by Next Generation Sequencing in Bacillus licheniformis DSM13

Prophages are viruses, which have integrated their genomes into the genome of a bacterial host. The status of the prophage genome can vary from fully intact with the potential to form infective particles to a remnant state where only a few phage genes persist. Prophages have impact on the properties of their host and are therefore of great interest for genomic research and strain design. Here we present a genome- and next generation sequencing (NGS)-based approach for identification and activity evaluation of prophage regions. Seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Analysis of isolated phage particles (induced by mitomycin C) from the wild-type strain and prophage deletion mutant strains revealed activity of the prophage regions BLi_Pp2 (PBSX-like), BLi_Pp3 and BLi_Pp6. In contrast to BLi_Pp2 and BLi_Pp3, neither phage DNA nor phage particles of BLi_Pp6 could be visualized. However, the ability of prophage BLi_Pp6 to generate particles could be confirmed by sequencing of particle-protected DNA mapping to prophage locus BLi_Pp6. The introduced NGS-based approach allows the investigation of prophage regions and their ability to form particles. Our results show that this approach increases the sensitivity of prophage activity analysis and can complement more conventional approaches such as transmission electron microscopy (TEM).     

A GFP-Tagged Gross Deletion on Chromosome 1 Causes Malignant Peripheral Nerve Sheath Tumors and Carcinomas in Zebrafish

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas, characterized by complex karyotypes. The molecular bases of such malignancy are poorly understood and efficient targeted molecular therapies are currently lacking. Here we describe a novel zebrafish model of MPNSTs, represented by the transgenic mutant line Tg(-8.5nkx2.2a:GFP) ^ia2. ia2 homozygous animals displayed embryonic lethality by 72 hpf, while the heterozygotes develop visible tumor masses with high frequency in adulthood. Histological and immunohistochemical examination revealed aggressive tumors with either mesenchymal or epithelial features. The former (54% of the cases) arose either in the abdominal cavity, or as intrathecal/intraspinal lesions and is composed of cytokeratin-negative spindle cells with fascicular/storiform growth pattern consistent with zebrafish MPNSTs. The second histotype was composed by polygonal or elongated cells, immunohistochemically positive for the pan-cytokeratin AE1/AE3. The overall histologic and immunohistochemical features were consistent with a malignant epithelial neoplasm of possible gastrointestinal/pancreatic origin. With an integrated approach, based on microsatellite (VNTR) and STS markers, we showed that ia2 insertion, in Tg(-8.5nkx2.2a:GFP) ^ia2 embryos, is associated with a deletion of 15.2 Mb in the telomeric portion of chromosome 1. Interestingly, among ia2 deleted genes we identified the presence of the 40S ribosomal protein S6 gene that may be one of the possible drivers for the MPNSTs in ia2 mutants. Thanks to the peculiar features of zebrafish as animal model of human cancer (cellular and genomic similarity, transparency and prolificacy) and the GFP tag, the Tg(-8.5nkx2.2a:GFP) ^ia2 line provides a manageable tool to study in vivo with high frequency MPNST biology and genetics, and to identify, in concert with the existing zebrafish MPNST models, conserved relevant mechanisms in zebrafish and human cancer development.     

Infant Growth after Preterm Birth and Mental Health in Young Adulthood

Objectives

Faster growth after preterm birth benefits long-term cognitive functioning. Whether these benefits extend to mental health remains largely unknown. We examined if faster growth in infancy is associated with better self-reported mental health in young adults born preterm at very low birth weight (VLBW) (<1500g).

Study Design

As young adults, participants of the Helsinki Study of Very Low Birth Weight Adults self-reported symptoms of depression and attention deficit/hyperactivity disorder (ADHD) (n = 157) and other psychiatric problems (n = 104). As main predictors of mental health outcomes in linear regression models, we used infant weight, length, and head circumference at birth, term, and 12 months of corrected age, and growth between these time points. Growth data were collected from records and measures at term and at 12 months of corrected age were interpolated. Additionally, we examined the moderating effects of intrauterine growth restriction.

Results

Size at birth, term, or 12 months of corrected age, or growth between these time points were not associated with mental health outcomes (p-values >0.05). Intrauterine growth restriction did not systematically moderate any associations.

Conclusions

Despite the high variability in early growth of VLBW infants, the previously described association between slow growth in infancy and poorer cognitive functioning in later life is not reflected in symptoms of depression, ADHD, and other psychiatric problems. This suggests that the development of cognitive and psychiatric problems may have dissimilar critical periods in VLBW infants.     

Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing’s Sarcoma

Ewing’s sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing’s sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing’s sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing’s sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing’s sarcoma patients with PARP inhibitors.