Methodology for reliable and reproducible cryopreservation of human cervical tissue

BACKGROUND: In order to conduct laboratory studies on donated cervical tissue at suitable times an effective and reliable cryopreservation protocol for cervical tissue is required. METHODS: An active freezing approach was devised utilising 10% dimethyl sulfoxide in foetal bovine serum as a cryoprotective agent with a cooling rate of 1 °C/min to -50 °C then 10 °C/min to -120 °C; a related thawing protocol was also optimised which would allow for the bio-banking of cervical tissue. Viability of freshly harvested cervical tissue was compared to frozen-thawed samples utilising colorimetric MTT assay. In parallel, fresh and freeze-thawed samples were cultured and tested on days 1, 7 and 14 to determine whether bio-banking had detrimental effects on tissue viability over time. RESULTS: Repeat testing revealed that tissue viability between fresh and freeze-thawed wasand freeze-thawed samples was comparable at all four time points (days 0, 1, 7 and 14) with no apparent reductions of viability, thus demonstrating this method of cryopreserving cervical tissue is reliable and reproducible, without detrimental effects on live tissue culture. We believe this methodology creates the opportunity for bio-banking donated cervical tissues, which aids improved experimental design and reduces time pressures and wastage

Cell-specific effects of Nox2 on the acute and chronic response to myocardial infarction

BACKGROUND: Increased reactive oxygen species (ROS) production is involved in the process of adverse cardiac remodeling and development of heart failure after myocardial infarction (MI). NADPH oxidase-2 (Nox2) is a major ROS source within the heart and its activity increases after MI. Furthermore, genetic deletion of Nox2 is protective against post-MI cardiac remodeling. Nox2 levels may increase both in cardiomyocytes and endothelial cells and recent studies indicate cell-specific effects of Nox2, but it is not known which of these cell types is important in post-MI remodeling. METHODS AND RESULTS: We have generated transgenic mouse models in which Nox2 expression is targeted either to cardiomyocytes (cardio-Nox2TG) or endothelial cells (endo-Nox2TG). We here studied the response of cardio-Nox2TG mice, endo-Nox2TG mice and matched wild-type littermates (WT) to MI induced by permanent left coronary artery ligation up to 4weeks. Initial infarct size assessed by magnetic resonance imaging (MRI) and cardiac dysfunction were similar among groups. Cardiomyocyte hypertrophy and interstitial fibrosis were augmented in cardio-Nox2TG compared to WT after MI and post-MI survival tended to be worse whereas endo-Nox2TG mice showed no significant difference compared to WT. CONCLUSIONS: These results indicate that cardiomyocyte rather than endothelial cell Nox2 may have the more important role in post-MI remodeling

Gay and Bisexual Men's Willingness to Receive Anal Papanicolaou Testing

Objectives. We assessed the willingness of gay and bisexual men, who have high rates of anal cancer that might be prevented through regular screening, to receive anal Papanicolaou tests

Contractile Function during Angiotensin-II Activation:Increased Nox2 Activity Modulates Cardiac Calcium Handling via Phospholamban Phosphorylation

AbstractBackgroundRenin-angiotensin system activation is a feature of many cardiovascular conditions. Activity of myocardial reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (NADPH oxidase 2 or Nox2) is enhanced by angiotensin II (Ang II) and contributes to increased hypertrophy, fibrosis, and adverse remodeling. Recent studies found that Nox2-mediated reactive oxygen species production modulates physiological cardiomyocyte function.ObjectivesThis study sought to investigate the effects of cardiomyocyte Nox2 on contractile function during increased Ang II activation.MethodsWe generated a cardiomyocyte-targeted Nox2-transgenic mouse model and studied the effects of in vivo and ex vivo Ang II stimulation, as well as chronic aortic banding.ResultsChronic subpressor Ang II infusion induced greater cardiac hypertrophy in transgenic than wild-type mice but unexpectedly enhanced contractile function. Acute Ang II treatment also enhanced contractile function in transgenic hearts in vivo and transgenic cardiomyocytes ex vivo. Ang II–stimulated Nox2 activity increased sarcoplasmic reticulum (SR) Ca2+ uptake in transgenic mice, increased the Ca2+ transient and contractile amplitude, and accelerated cardiomyocyte contraction and relaxation. Elevated Nox2 activity increased phospholamban phosphorylation in both hearts and cardiomyocytes, related to inhibition of protein phosphatase 1 activity. In a model of aortic banding–induced chronic pressure overload, heart function was similarly depressed in transgenic and wild-type mice.ConclusionsWe identified a novel mechanism in which Nox2 modulates cardiomyocyte SR Ca2+ uptake and contractile function through redox-regulated changes in phospholamban phosphorylation. This mechanism can drive increased contractility in the short term in disease states characterized by enhanced renin-angiotensin system activation

Validating a prediction tool to determine the risk of nosocomial multidrug-resistant Gram-negative bacilli infection in critically ill patients: A retrospective case–control study

BACKGROUND:The Singapore GSDCS score was developed to enable clinicians predict the risk of nosocomial multidrug-resistant Gram-negative bacilli (RGNB) infection in critically ill patients. We aimed to validate this score in a UK setting. METHOD:A retrospective case-control study was conducted including patients who stayed for more than 24h in intensive care units (ICUs) across two tertiary National Health Service hospitals in London, UK (April 2011-April 2016). Cases with RGNB and controls with sensitive Gram-negative bacilli (SGNB) infection were identified. RESULTS:The derived GSDCS score was calculated from when there was a step change in antimicrobial therapy in response to clinical suspicion of infection as follows: prior Gram-negative organism, Surgery, Dialysis with end-stage renal disease, prior Carbapenem use and intensive care Stay of more than 5 days. A total of 110 patients with RGNB infection (cases) were matched 1:1 to 110 geotemporally chosen patients with SGNB infection (controls). The discriminatory ability of the prediction tool by receiver operating characteristic curve analysis in our validation cohort was 0.75 (95% confidence interval 0.65-0.81), which is comparable with the area under the curve of the derivation cohort (0.77). The GSDCS score differentiated between low- (0-1.3), medium- (1.4-2.3) and high-risk (2.4-4.3) patients for RGNB infection (P<0.001) in a UK setting. CONCLUSION:A simple bedside clinical prediction tool may be used to identify and differentiate patients at low, medium and high risk of RGNB infection prior to initiation of prompt empirical antimicrobial therapy in the intensive care setting

Endothelial NADPH oxidase-2 promotes interstitial cardiac fibrosis and diastolic dysfunction through proinflammatory effects and endothelial-mesenchymal transition

OBJECTIVES: This study sought to investigate the effect of endothelial dysfunction on the development of cardiac hypertrophy and fibrosis. BACKGROUND: Endothelial dysfunction accompanies cardiac hypertrophy and fibrosis, but its contribution to these conditions is unclear. Increased nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) activation causes endothelial dysfunction. METHODS: Transgenic mice with endothelial-specific NOX2 overexpression (TG mice) and wild-type littermates received long-term angiotensin II (AngII) infusion (1.1 mg/kg/day, 2 weeks) to induce hypertrophy and fibrosis. RESULTS: TG mice had systolic hypertension and hypertrophy similar to those seen in wild-type mice but developed greater cardiac fibrosis and evidence of isolated left ventricular diastolic dysfunction (p < 0.05). TG myocardium had more inflammatory cells and VCAM-1-positive vessels than did wild-type myocardium after AngII treatment (both p < 0.05). TG microvascular endothelial cells (ECs) treated with AngII recruited 2-fold more leukocytes than did wild-type ECs in an in vitro adhesion assay (p < 0.05). However, inflammatory cell NOX2 per se was not essential for the profibrotic effects of AngII. TG showed a higher level of endothelial-mesenchymal transition (EMT) than did wild-type mice after AngII infusion. In cultured ECs treated with AngII, NOX2 enhanced EMT as assessed by the relative expression of fibroblast versus endothelial-specific markers. CONCLUSIONS: AngII-induced endothelial NOX2 activation has profound profibrotic effects in the heart in vivo that lead to a diastolic dysfunction phenotype. Endothelial NOX2 enhances EMT and has proinflammatory effects. This may be an important mechanism underlying cardiac fibrosis and diastolic dysfunction during increased renin-angiotensin activation

Nox4 reprograms cardiac substrate metabolism via protein O-GlcNAcylation to enhance stress adaptation.

Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4-dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial

Distinct regulatory effects of myeloid cell and endothelial cell Nox2 on blood pressure

Background -Hypertension due to increased renin angiotensin system (RAS) activation is associated with elevated reactive oxygen species (ROS) production. Previous studies implicate NADPH oxidase (Nox) proteins as important ROS sources during RAS activation, with different Nox isoforms being potentially involved. Among these, Nox2 is expressed in multiple cell types including endothelial cells, fibroblasts, immune cells and microglia. Blood pressure (BP) is regulated at central nervous system, renal and vascular levels but the cell-specific role of Nox2 in BP regulation is unknown. Methods -We generated a novel mouse model with a Floxed Nox2 gene and used Tie2-Cre, LysM Cre or Cdh5-CreERT2 driver lines to develop cell-specific models of Nox2 perturbation to investigate its role in BP regulation. Results -Unexpectedly, Nox2 deletion in myeloid but not endothelial cells resulted in a significant reduction in basal BP. Tie2-CreNox2 knockout (KO) mice (in which Nox2 was deficient in both endothelial cells and myeloid cells) and LysM Cre Nox2KO mice (in which Nox2 was deficient in myeloid cells) both had significantly lower BP than littermate controls whereas basal BP was unaltered in Cdh5-CreERT2 Nox2 KO mice (in which Nox2 is deficient only in endothelial cells). The lower BP was attributable to an increased NO bioavailability which dynamically dilated resistance vessels in vivo under basal conditions, without change in renal function. Myeloid-specific Nox2 deletion had no effect on angiotensin II-induced hypertension which, however, was blunted in Tie2-CreNox2KO mice along with preservation of endothelium-dependent relaxation during angiotensin II stimulation. Conclusions -We identify a hitherto unrecognized modulation of basal BP by myeloid cell Nox2 whereas endothelial cell Nox2 regulates angiotensin II-induced hypertension. These results identify distinct cell-specific roles for Nox2 in BP regulation

The dynamics of risk perceptions and precautionary behavior in response to 2009 (H1N1) pandemic influenza

<p>Abstract</p> <p>Background</p> <p>The trajectory of an infectious disease outbreak is affected by the behavior of individuals, and the behavior is often related to individuals' risk perception. We assessed temporal changes and geographical differences in risk perceptions and precautionary behaviors in response to H1N1 influenza.</p> <p>Methods</p> <p>1,290 US adults completed an online survey on risk perceptions, interests in pharmaceutical interventions (preventive intervention and curative intervention), and engagement in precautionary activities (information seeking activities and taking quarantine measures) in response to H1N1 influenza between April 28 and May 27 2009. Associations of risk perceptions and precautionary behaviors with respondents' sex, age, and household size were analyzed. Linear and quadratic time trends were assessed by regression analyses. Geographic differences in risk perception and precautionary behaviors were evaluated. Predictors of willingness to take pharmaceutical intervention were analyzed.</p> <p>Results</p> <p>Respondents from larger households reported stronger interest in taking medications and engaged in more precautionary activities, as would be normatively predicted. Perceived risk increased over time, whereas interest in pharmaceutical preventive interventions and the engagement in some precautionary activities decreased over time. Respondents who live in states with higher H1N1 incidence per population perceived a higher likelihood of influenza infection, but did not express greater interests in pharmaceutical interventions, nor did they engage in a higher degree of precautionary activities. Perceived likelihood of influenza infection, willingness to take medications and engagement in information seeking activities were higher for women than men.</p> <p>Conclusions</p> <p>Perceived risk of infection and precautionary behavior can be dynamic in time, and differ by demographic characteristics and geographical locations. These patterns will likely influence the effectiveness of disease control measures.</p

Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2a-mediated stress signaling

Source: doi: 10.15252/embj.201592394Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine–threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species‐generating NADPH oxidase‐4 (Nox4) is induced downstream of ATF4, binds to a PP1‐targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4‐regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia–reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4–GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress