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1.  Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8 
Journal of Virology  2014;88(2):799-810.
RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5′-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.
doi:10.1128/JVI.01571-13
PMCID: PMC3911656  PMID: 24227841
2.  Distinctive Roles for OAS and PKR in the in Vivo Anti-Viral Effect of an Adenoviral Vector Expressing Murine IFN-β1 
To evaluate the anti-herpes simplex virus type 1 (HSV-1) mechanisms of murine IFN-β in ocular infection mice were transduced with an adenoviral vector expressing murine IFN-β (Ad:IFN-β). Ocular transduction with Ad:IFN-β resulted in enhanced survival following infection with HSV-1. The protective effect was associated with a reduction in (i) viral titer, (ii) viral gene expression, (iii) IFN-γ levels, and (iv) the percentage of CD8+ T lymphocyte and NK cell infiltration in infected tissue. Expression of IFN-β resulted in an elevation of the IFN-induced anti-viral gene 2′ 5′-oligoadenylate synthetase (OAS1a) but not double-stranded RNA-dependent protein kinase (PKR) in the cornea and trigeminal ganglion (TG). Mice deficient in the downstream effector molecule of the OAS pathway, RNase L, were no more sensitive to ocular HSV-1 compared to wild type controls in the TG measuring viral titer. However, the efficacy of Ad:IFN-β was transiently lost in the eyes of RNase L mice. By comparison, PKR deficient mice were more susceptible to ocular HSV-1 infection and the anti-viral efficacy following transduction with Ad:IFN-β was significantly diminished in the eye and TG. These results suggest that PKR is central in controlling ocular HSV-1 infection in the absence of exogenous IFN whereas the OAS pathway appears to respond to exogenous IFN contributing to the establishment of an anti-viral environment in a tissue-restricted manner.
PMCID: PMC4060620  PMID: 15100308
HSV-1; keratitis; adenoviral vector; IFN-β; OAS; OKR
3.  Patient self-monitoring of blood pressure and self-titration of medication in primary care: the TASMINH2 trial qualitative study of health professionals’ experiences 
The British Journal of General Practice  2013;63(611):e378-e385.
Background
Self-monitoring with self-titration of antihypertensives leads to reduced blood pressure. Patients are keen on self-monitoring but little is known about healthcare professional views.
Aim
To explore health professionals‘ views and experiences of patient self-management, particularly with respect to future implementation into routine care.
Design and setting
Qualitative study embedded within a randomised controlled trial of healthcare professionals participating in the TASMINH2 trial of patient self-monitoring with self-titration of antihypertensives from 24 West Midlands general practices.
Method
Taped and transcribed semi-structured interviews with 13 GPs, two practice nurses and one healthcare assistant. Constant comparative method of analysis.
Results
Primary care professionals were positive about self-monitoring, but procedures for ensuring patients measured blood pressure correctly were haphazard. GPs interpreted home readings variably, with many not making adjustment for lower home blood pressure. Interviewees were satisfied with patient training and arrangements for blood pressure monitoring and self-titration of medication during the trial, but less sure about future implementation into routine care. There was evidence of a need for training of both patients and professionals for successful integration of self-management.
Conclusion
Health professionals wanted more patient involvement in hypertension care but needed a framework to work within. Consideration of how to train patients to measure blood pressure and how home readings become part of their care is required before self-monitoring and self-titration can be implemented widely. As home monitoring becomes more widespread, the development of patient self-management, including self-titration of medication, should follow but this may take time to achieve.
doi:10.3399/bjgp13X668168
PMCID: PMC3662454  PMID: 23735408
general practice; hypertension, primary care; qualitative research, self-monitoring
4.  Blood pressure and incidence of twelve cardiovascular diseases: lifetime risks, healthy life-years lost, and age-specific associations in 1·25 million people 
Lancet  2014;383(9932):1899-1911.
Summary
Background
The associations of blood pressure with the different manifestations of incident cardiovascular disease in a contemporary population have not been compared. In this study, we aimed to analyse the associations of blood pressure with 12 different presentations of cardiovascular disease.
Methods
We used linked electronic health records from 1997 to 2010 in the CALIBER (CArdiovascular research using LInked Bespoke studies and Electronic health Records) programme to assemble a cohort of 1·25 million patients, 30 years of age or older and initially free from cardiovascular disease, a fifth of whom received blood pressure-lowering treatments. We studied the heterogeneity in the age-specific associations of clinically measured blood pressure with 12 acute and chronic cardiovascular diseases, and estimated the lifetime risks (up to 95 years of age) and cardiovascular disease-free life-years lost adjusted for other risk factors at index ages 30, 60, and 80 years. This study is registered at ClinicalTrials.gov, number NCT01164371.
Findings
During 5·2 years median follow-up, we recorded 83 098 initial cardiovascular disease presentations. In each age group, the lowest risk for cardiovascular disease was in people with systolic blood pressure of 90–114 mm Hg and diastolic blood pressure of 60–74 mm Hg, with no evidence of a J-shaped increased risk at lower blood pressures. The effect of high blood pressure varied by cardiovascular disease endpoint, from strongly positive to no effect. Associations with high systolic blood pressure were strongest for intracerebral haemorrhage (hazard ratio 1·44 [95% CI 1·32–1·58]), subarachnoid haemorrhage (1·43 [1·25–1·63]), and stable angina (1·41 [1·36–1·46]), and weakest for abdominal aortic aneurysm (1·08 [1·00–1·17]). Compared with diastolic blood pressure, raised systolic blood pressure had a greater effect on angina, myocardial infarction, and peripheral arterial disease, whereas raised diastolic blood pressure had a greater effect on abdominal aortic aneurysm than did raised systolic pressure. Pulse pressure associations were inverse for abdominal aortic aneurysm (HR per 10 mm Hg 0·91 [95% CI 0·86–0·98]) and strongest for peripheral arterial disease (1·23 [1·20–1·27]). People with hypertension (blood pressure ≥140/90 mm Hg or those receiving blood pressure-lowering drugs) had a lifetime risk of overall cardiovascular disease at 30 years of age of 63·3% (95% CI 62·9–63·8) compared with 46·1% (45·5–46·8) for those with normal blood pressure, and developed cardiovascular disease 5·0 years earlier (95% CI 4·8–5·2). Stable and unstable angina accounted for most (43%) of the cardiovascular disease-free years of life lost associated with hypertension from index age 30 years, whereas heart failure and stable angina accounted for the largest proportion (19% each) of years of life lost from index age 80 years.
Interpretation
The widely held assumptions that blood pressure has strong associations with the occurrence of all cardiovascular diseases across a wide age range, and that diastolic and systolic associations are concordant, are not supported by the findings of this high-resolution study. Despite modern treatments, the lifetime burden of hypertension is substantial. These findings emphasise the need for new blood pressure-lowering strategies, and will help to inform the design of randomised trials to assess them.
Funding
Medical Research Council, National Institute for Health Research, and Wellcome Trust.
doi:10.1016/S0140-6736(14)60685-1
PMCID: PMC4042017  PMID: 24881994
5.  Parental Human Papillomavirus Vaccine Survey (PHPVS): Nurse-Led Instrument Development and Psychometric Testing for Use in Research and Primary Care Screening 
Journal of nursing measurement  2013;21(1):96-109.
Background and Purpose
Human papillomavirus (HPV) vaccine was approved for girls aged 9–24 years in 2006 to prevent HPV infection and cervical cancer. The Parental Human Papillomavirus Survey (PHPVS) was framed on theoretical constructs of the health belief model (HBM) and developed to survey parents regarding their HPV knowledge, attitudes, and intent to vaccinate.
Methods
We evaluated the psychometric properties of the PHPVS using classical item analysis and exploratory factor analysis (EFA) among a sample of 200 parents/caregivers.
Results
The EFA yielded a 4-factor unidimensional model that explained between 62% and 68% of the total variance depending on the extraction method used. The estimated Cronbach’s alpha for the PHPVS was .96.
Conclusions
The PHPVS is a reliable measure of HPV knowledge, attitudes, and intent to vaccinate.
PMCID: PMC3952023  PMID: 23786137
HPV vaccines; health surveys; health care disparities; pediatrics; adolescents; Parents
6.  Rationale and study design of the Prospective comparison of Angiotensin Receptor neprilysin inhibitor with Angiotensin receptor blocker MEasuring arterial sTiffness in the eldERly (PARAMETER) study 
BMJ Open  2014;4(2):e004254.
Introduction
Hypertension in elderly people is characterised by elevated systolic blood pressure (SBP) and increased pulse pressure (PP), which indicate large artery ageing and stiffness. LCZ696, a first-in-class angiotensin receptor neprilysin inhibitor (ARNI), is being developed to treat hypertension and heart failure. The Prospective comparison of Angiotensin Receptor neprilysin inhibitor with Angiotensin receptor blocker MEasuring arterial sTiffness in the eldERly (PARAMETER) study will assess the efficacy of LCZ696 versus olmesartan on aortic stiffness and central aortic haemodynamics.
Methods and analysis
In this 52-week multicentre study, patients with hypertension aged ≥60 years with a mean sitting (ms) SBP ≥150 to <180 and a PP>60 mm Hg will be randomised to once daily LCZ696 200 mg or olmesartan 20 mg for 4 weeks, followed by a forced-titration to double the initial doses for the next 8 weeks. At 12–24 weeks, if the BP target has not been attained (msSBP <140  and ms diastolic BP <90 mm Hg), amlodipine (2.5–5 mg) and subsequently hydrochlorothiazide (6.25–25 mg) can be added. The primary and secondary endpoints are changes from baseline in central aortic systolic pressure (CASP) and central aortic PP (CAPP) at week 12, respectively. Other secondary endpoints are the changes in CASP and CAPP at week 52. A sample size of 432 randomised patients is estimated to ensure a power of 90% to assess the superiority of LCZ696 over olmesartan at week 12 in the change from baseline of mean CASP, assuming an SD of 19 mm Hg, the difference of 6.5 mm Hg and a 15% dropout rate. The primary variable will be analysed using a two-way analysis of covariance.
Ethics and dissemination
The study was initiated in December 2012 and final results are expected in 2015. The results of this study will impact the design of future phase III studies assessing cardiovascular protection.
Clinical trials identifier
EUDract number 2012-002899-14 and ClinicalTrials.gov NCT01692301.
doi:10.1136/bmjopen-2013-004254
PMCID: PMC3918996  PMID: 24496699
7.  Rationale and design of the PRognostic Importance of MIcrovascular Dysfunction in asymptomatic patients with Aortic Stenosis (PRIMID-AS): a multicentre observational study with blinded investigations 
BMJ Open  2013;3(12):e004348.
Introduction
Aortic stenosis (AS) is the commonest valve disorder in the developed world requiring surgery. Surgery in patients with severe asymptomatic AS remains controversial. Exercise testing can identify asymptomatic patients at increased risk of death and symptom development, but with limited specificity, especially in older adults. Cardiac MRI (CMR), including myocardial perfusion reserve (MPR) may be a novel imaging biomarker in AS.
Aims
(1) To improve risk stratification in asymptomatic patients with AS and (2) to determine whether MPR is a better predictor of outcome than exercise testing and brain natriuretic peptide (BNP).
Method/design
Multicentre, prospective observational study in the UK, comparing MPR with exercise testing and BNP (with blinded CMR analysis) for predicting outcome.
Population
170 asymptomatic patients with moderate-to-severe AS, who would be considered for aortic valve replacement (AVR).
Primary outcome
Composite of: typical symptoms necessitating referral for AVR and major adverse cardiovascular events. Follow-up: 12–30 months (minimum 12 months).
Primary hypothesis
MPR will be a better predictor of outcome than exercise testing and BNP.
Ethics/dissemination
The study has full ethical approval and is actively recruiting patients. Data collection will be completed in November 2014 and the study results will be submitted for publication within 6 months of completion.
ClinicalTrials.gov identifier
NCT01658345.
doi:10.1136/bmjopen-2013-004348
PMCID: PMC3884636  PMID: 24353258
8.  Structure and function of OprD protein in Pseudomonas aeruginosa: From antibiotic resistance to novel therapies 
International journal of medical microbiology : IJMM  2012;302(2):10.1016/j.ijmm.2011.10.001.
Pseudomonas aeruginosa (P. aeruginosa) is a common pathogen isolated from patients with nosocomial infections. Due to its intrinsic and acquired antimicrobial resistance, limited classes of antibiotics can be used for the treatment of infection with P. aeruginosa. Of these, the carbapenems are very important; however, the occurrence of carbapenem-resistant strains is gradually increasing over time. Deficiency of the outer membrane protein OprD confers P. aeruginosa a basal level of resistance to carbapenems, especially to imipenem. Functional studies have revealed that loops 2 and 3 in the OprD protein contain the entrance and/or binding sites for imipenem. Therefore, any mutation in loop 2 and/or loop 3 that causes conformational changes could result in carbapenem resistance. OprD is also a common channel for some amino acids and peptides, and competition with carbapenems through the channel may also occur. Furthermore, OprD is a highly regulated protein at transcriptional and post-transcriptional levels by some metals, small bioactive molecules, amino acids, and efflux pump regulators. Because of its hypermutability and highly regulated properties, OprD is thought to be the most prevalent mechanism for carbapenem resistance in P. aeruginosa. Developing new strategies to combat infection with carbapenem-resistant P. aeruginosa lacking OprD is an ongoing challenge.
doi:10.1016/j.ijmm.2011.10.001
PMCID: PMC3831278  PMID: 22226846
Pseudomonas aeruginosa; OprD; Carbapenem
9.  24-hour central aortic systolic pressure and 24-hour central pulse pressure are related to diabetic complications in type 1 diabetes – a cross-sectional study 
Background
Non-invasive measurements of 24 hour ambulatory central aortic systolic pressure (24 h-CASP) and central pulse pressure (24 h-CPP) are now feasible. We evaluate the relationship between 24 h central blood pressure and diabetes-related complications in patients with type 1 diabetes.
Methods
The study was cross-sectional, including 715 subjects: 86 controls (C), 69 patients with short diabetes duration (< 10 years), normoalbuminuria (< 30 mg/24 h) without receiving antihypertensive treatment (SN), 211 with longstanding diabetes (≥ 10 years) and normoalbuminuria (LN), 163 with microalbuminuria (30-299 mg/24 h) (Mi) and 186 with macroalbuminuria (> 300 mg/24 h) (Ma).
24 h-CASP and 24 h-CPP was measured using a tonometric wrist-watch-like device (BPro, HealthStats, Singapore) and derived using N-point moving average.
Results
In C, SN, LN, Mi and Ma mean ± SD 24 h-CASP was: 114 ± 17, 115 ± 13, 121 ± 13, 119 ± 16 and 121 ± 13 mmHg (p < 0.001); and 24 h-CPP: 38 ± 8, 38 ± 7, 44 ± 10, 46 ± 11 and 46 ± 11 mmHg, (p < 0.001).
Following rigorous adjustment (24 h mean arterial pressure and conventional risk factors), 24 h-CASP and 24 h-CPP increased with diabetes, albuminuria degree, previous cardiovascular disease (CVD), retinopathy and autonomic dysfunction (p ≤ 0.031).
Odds ratios per 1 standard deviation increase in 24 h-CASP, 24 h-CPP and 24 h systolic blood pressure (24 h-SBP) were for CVD: 3.19 (1.68-6.05), 1.43 (1.01-2.02) and 2.39 (1.32-4.33), retinopathy: 4.41 (2.03-9.57), 1.77 (1.17-2.68) and 3.72 (1.85-7.47) and autonomic dysfunction: 3.25 (1.65-6.41), 1.64 (1.12-2.39) and 2.89 (1.54-5.42).
Conclusions
24 h-CASP and 24 h-CPP was higher in patients vs. controls and increased with diabetic complications independently of covariates. Furthermore, 24 h-CASP was stronger associated to complications than 24 h-SBP.
The prognostic significance of 24 h-CASP and 24 h-CPP needs to be determined in follow-up studies.
Trial registration
ClinicalTrials.gov ID NCT01171248.
doi:10.1186/1475-2840-12-122
PMCID: PMC3765827  PMID: 23978271
Ambulatory blood pressure; Brachial systolic blood pressure; Central aortic systolic pressure; Central pulse pressure; Diabetic complications; Type 1 diabetes
10.  A role for mitochondrial oxidants in stress-induced premature senescence of human vascular smooth muscle cells☆ 
Redox Biology  2013;1(1):411-417.
Mitochondria are a major source of cellular oxidants and have been implicated in aging and associated pathologies, notably cardiovascular diseases. Vascular cell senescence is observed in experimental and human cardiovascular pathologies. Our previous data highlighted a role for angiotensin II in the induction of telomere-dependent and -independent premature senescence of human vascular smooth muscle cells and suggested this was due to production of superoxide by NADPH oxidase. However, since a role for mitochondrial oxidants was not ruled out we hypothesise that angiotensin II mediates senescence by mitochondrial superoxide generation and suggest that inhibition of superoxide may prevent vascular smooth muscle cell aging in vitro. Cellular senescence was induced using a stress-induced premature senescence protocol consisting of three successive once-daily exposure of cells to 1×10−8 mol/L angiotensin II and was dependent upon the type-1 angiotensin II receptor. Angiotensin stimulated NADPH-dependent superoxide production as estimated using lucigenin chemiluminescence in cell lysates and this was attenuated by the mitochondrial electron transport chain inhibitor, rotenone. Angiotensin also resulted in an increase in mitoSOX fluorescence indicating stimulation of mitochondrial superoxide. Significantly, the induction of senescence by angiotensin II was abrogated by rotenone and by the mitochondria-targeted superoxide dismutase mimetic, mitoTEMPO. These data suggest that mitochondrial superoxide is necessary for the induction of stress-induced premature senescence by angiotensin II and taken together with other data suggest that mitochondrial cross-talk with NADPH oxidases, via as yet unidentified signalling pathways, is likely to play a key role.
Graphical abstract
Highlights
•Angiotensin II causes stress-induced premature senescence in hVSMC.•Mitochondrial superoxide is necessary for premature senescence.•Mitochondrial cross-talk with NADPH oxidases is implicated in this mechanism.
doi:10.1016/j.redox.2013.08.004
PMCID: PMC3814954  PMID: 24191234
Vascular smooth muscle cell; Angiotensin II; Mitochondria; Superoxide; Cell senescence; Stress-induced premature senescence
11.  Targets and self-management for the control of blood pressure in stroke and at risk groups (TASMIN-SR): protocol for a randomised controlled trial 
Background
Self-monitoring of hypertension with self-titration of antihypertensives (self-management) results in lower systolic blood pressure for at least one year. However, few people in high risk groups have been evaluated to date and previous work suggests a smaller effect size in these groups. This trial therefore aims to assess the added value of self-management in high risk groups over and above usual care.
Methods/Design
The targets and self-management for the control of blood pressure in stroke and at risk groups (TASMIN-SR) trial will be a pragmatic primary care based, unblinded, randomised controlled trial of self-management of blood pressure (BP) compared to usual care. Eligible patients will have a history of stroke, coronary heart disease, diabetes or chronic kidney disease and will be recruited from primary care. Participants will be individually randomised to either usual care or self-management. The primary outcome of the trial will be difference in office SBP between intervention and control groups at 12 months adjusted for baseline SBP and covariates. 540 patients will be sufficient to detect a difference in SBP between self-management and usual care of 5 mmHg with 90% power. Secondary outcomes will include self-efficacy, lifestyle behaviours, health-related quality of life and adverse events. An economic analysis will consider both within trial costs and a model extrapolating the results thereafter. A qualitative analysis will gain insights into patients’ views, experiences and decision making processes.
Discussion
The results of the trial will be directly applicable to primary care in the UK. If successful, self-management of blood pressure in people with stroke and other high risk conditions would be applicable to many hundreds of thousands of individuals in the UK and beyond.
Trial Registration
ISRCTN87171227
doi:10.1186/1471-2261-13-21
PMCID: PMC3623796  PMID: 23522245
Hypertension; Self-management; Stroke, Diabetes; Coronary heart disease; Chronic kidney disease; Primary care
12.  Patients’ experiences of self-monitoring blood pressure and self-titration of medication: the TASMINH2 trial qualitative study 
The British Journal of General Practice  2011;62(595):e135-e142.
Background
Self-management of hypertension, comprising self-monitoring of blood pressure with self-titration of medication, improves blood pressure control, but little is known regarding the views of patients undertaking it.
Aim
To explore patients’ views of self-monitoring blood pressure and self-titration of antihypertensive medication.
Design and setting
Qualitative study embedded within the randomised controlled trial TASMINH2 (Telemonitoirng and Self Management in the Control of Hypertension) trial of patient self-management of hypertension from 24 general practices in the West Midlands.
Method
Taped and transcribed semi-structured interviews with 23 intervention patients were used. Six family members were also interviewed. Analysis was by a constant comparative method.
Results
Patients were confident about self-monitoring and many felt their multiple home readings were more valid than single office readings taken by their GP. Although many patients self-titrated medication when required, others lacked the confidence to increase medication without reconsulting with their GP. Patients were more comfortable with titrating medication if their blood pressure readings were substantially above target, but were reluctant to implement such a change if readings were borderline. Many planned to continue self-monitoring after the study finished and report home readings to their GP, but few wished to continue with a self-management plan.
Conclusion
Participants valued the additional information and many felt confident in both self-monitoring blood pressure and self-titrating medication. The reluctance to change medication for borderline readings suggests behaviour similar to the clinical inertia seen for physicians in analogous circumstances. Additional support for those lacking in confidence to implement prearranged medication changes may allow more patients to undertake self-management.
doi:10.3399/bjgp12X625201
PMCID: PMC3268493  PMID: 22520791
family practice; hypertension; qualitative research; self blood pressure monitoring
13.  Conformational rearrangements of RIG-I receptor on formation of a multiprotein:dsRNA assembly 
Nucleic Acids Research  2013;41(5):3436-3445.
The retinoic acid inducible gene-I (RIG-I)-like family of receptors is positioned at the front line of our innate cellular defence system. RIG-I detects and binds to foreign duplex RNA in the cytoplasm of both immune and non-immune cells, and initiates the induction of type I interferons and pro-inflammatory cytokines. The mechanism of RIG-I activation by double-stranded RNA (dsRNA) involves a molecular rearrangement proposed to expose the N-terminal pair of caspase activation recruitment domains, enabling an interaction with interferon-beta promoter stimulator 1 (IPS-1) and thereby initiating downstream signalling. dsRNA is particularly stimulatory when longer than 20 bp, potentially through allowing binding of more than one RIG-I molecule. Here, we characterize full-length RIG-I and RIG-I subdomains combined with a stimulatory 29mer dsRNA using multi-angle light scattering and size-exclusion chromatography–coupled small-angle X-ray scattering, to build up a molecular model of RIG-I before and after the formation of a 2:1 protein:dsRNA assembly. We report the small-angle X-ray scattering–derived solution structure of the human apo-RIG-I and observe that on binding of RIG-I to dsRNA in a 2:1 ratio, the complex becomes highly extended and flexible. Hence, here we present the first model of the fully activated oligomeric RIG-I.
doi:10.1093/nar/gks1477
PMCID: PMC3597671  PMID: 23325848
14.  Resting Heart Rate and the Risk of Microvascular Complications in Patients With Type 2 Diabetes Mellitus 
Background
A higher resting heart rate is associated with an increased probability of cardiovascular complications and premature death in patients with type 2 diabetes mellitus. The impact of heart rate on the risk of developing microvascular complications, such as diabetic retinopathy and nephropathy, is, however, unknown. The present study tests the hypothesis that a higher resting heart rate is associated with an increased incidence and a greater progression of microvascular complications in patients with type 2 diabetes mellitus.
Methods and Results
The relation between baseline resting heart rate and the development of a major microvascular event was examined in 11 140 patients who participated in the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) study. Major microvascular events were defined as a composite of new or worsening nephropathy or new or worsening retinopathy. Patients with a higher baseline heart rate were at increased risk of a new major microvascular complication during follow-up (adjusted hazard ratio: 1.13 per 10 beats per minute; 95% confidence interval: 1.07–1.20; P<0.001). The excess hazard was evident for both nephropathy (adjusted hazard ratio: 1.16 per 10 beats per minute; 95% confidence interval: 1.08–1.25) and retinopathy (adjusted hazard ratio: 1.11 per 10 beats per minute; 95% confidence interval: 1.02–1.21).
Conclusion
Patients with type 2 diabetes mellitus who have a higher resting heart rate experience a greater incidence of new-onset or progressive nephropathy and retinopathy.
Clinical Trial Registration
URL: http://www.clinicaltrials.gov. Unique identifier: NCT00145925. http://www.advance-trial.com/static/html/prehome/prehome.asp
doi:10.1161/JAHA.112.002832
PMCID: PMC3541618  PMID: 23316296
diabetes mellitus, type 2; heart rate; microcirculation
15.  A miR-19 regulon that controls NF-κB signaling 
Nucleic Acids Research  2012;40(16):8048-8058.
Fine-tuning of inflammatory responses by microRNAs (miRNAs) is complex, as they can both enhance and repress expression of pro-inflammatory mediators. In this study, we investigate inflammatory responses following global miRNA depletion, to better define the overall contribution of miRNAs to inflammation. We demonstrate that miRNAs positively regulate Toll-like receptor signaling using inducible Dicer1 deletion and global miRNA depletion. We establish an important contribution of miR-19b in this effect, which potentiates nuclear factor-κB (NF-κB) activity in human and mouse cells. Positive regulation of NF-κB signaling by miR-19b involves the coordinated suppression of a regulon of negative regulators of NF-κB signaling (including A20/Tnfaip3, Rnf11, Fbxl11/Kdm2a and Zbtb16). Transfection of miR-19b mimics exacerbated the inflammatory activation of rheumatoid arthritis primary fibroblast-like synoviocytes, demonstrating its physiological importance in the pathology of this disease. This study constitutes, to our knowledge, the first description of a miR-19 regulon that controls NF-κB signaling, and suggests that targeting this miRNA and linked family members could regulate the activity of NF-κB signaling in inflammation.
doi:10.1093/nar/gks521
PMCID: PMC3439911  PMID: 22684508
16.  A mechanical diagnosis and treatment (MDT) approach for a patient with discogenic low back pain and a relevant lateral component: a case report 
Patients with a lateral lumbar shift are often managed with a frontal plane, manual correction first described by McKenzie. However, there is a subset of patients that require a frontal plane correction who do not demonstrate a lateral shift. Both of these patient groups have what is termed a relevant lateral component (RLC). The subset of patients who have an RLC, without a shift, has received very little attention in the literature to date. This case report describes a patient with no shift who had a remarkable disc extrusion identified by magnetic resonance imaging (MRI). The patient responded nicely to a combination of frontal and transverse plane loading strategies in six treatments using the mechanical diagnosis and treatment (MDT) evaluation and treatment system. The case report is unique in the literature in that it describes a patient with no apparent frontal plane, lumbar deformity that had documented evidence of a large disc extrusion and who responded to a variation of McKenzie’s flexion–rotation mobilization. The centralization phenomenon was demonstrated immediately when a non-weight bearing, multiplanar, loading strategy was administered.
doi:10.1179/2042618610Y.0000000008
PMCID: PMC3172947  PMID: 22547922
Back pain; McKenzie; Mechanical diagnosis and treatment; Physical therapy; Relevant lateral component
17.  The Promyelocytic Leukemia Zinc Finger Protein: Two Decades of Molecular Oncology 
The promyelocytic leukemia zinc finger (PLZF) protein, also known as Zbtb16 or Zfp145, was first identified in a patient with acute promyelocytic leukemia, where a reciprocal chromosomal translocation t(11;17)(q23;q21) resulted in a fusion with the RARA gene encoding retinoic acid receptor alpha. The wild-type Zbtb16 gene encodes a transcription factor that belongs to the POK (POZ and Krüppel) family of transcriptional repressors. In addition to nine Krüppel-type sequence-specific zinc fingers, which make it a member of the Krüppel-like zinc finger protein family, the PLZF protein contains an N-terminal BTB/POZ domain and RD2 domain. PLZF has been shown to be involved in major developmental and biological processes, such as spermatogenesis, hind limb formation, hematopoiesis, and immune regulation. PLZF is localized mainly in the nucleus where it exerts its transcriptional repression function, and many post-translational modifications affect this ability and also have an impact on its cytoplasmic/nuclear dissociation. PLZF achieves its transcriptional regulation by binding to many secondary molecules to form large multi-protein complexes that bind to the regulatory elements in the promoter region of the target genes. These complexes are also capable of physically interacting with its target proteins. Recently, PLZF has become implicated in carcinogenesis as a tumor suppressor gene, since it regulates the cell cycle and apoptosis in many cell types. This review will examine the major advances in our knowledge of PLZF biological activities that augment its value as a therapeutic target, particularly in cancer and immunological diseases.
doi:10.3389/fonc.2012.00074
PMCID: PMC3398472  PMID: 22822476
PLZF; cancer; leukemia; cell cycle; stem cells; apoptosis; cytokines
18.  Urinary Concentrations of Dialkylphosphate Metabolites of Organophosphorus Pesticides: National Health and Nutrition Examination Survey 1999–2004 
Organophosphorus (OP) insecticides were among the first pesticides that EPA reevaluated as part of the Food Quality Protection Act of 1996. Our goal was to assess exposure to OP insecticides in the U.S. general population over a six-year period. We analyzed 7,456 urine samples collected as part of three two-year cycles of the National Health and Nutrition Examination Survey (NHANES) from 1999–2004. We measured six dialkylphosphate metabolites of OP pesticides to assess OP pesticide exposure. In NHANES 2003–2004, dimethylthiophosphate was detected most frequently with median and 95th percentile concentrations of 2.03 and 35.3 μg/L, respectively. Adolescents were two to three times more likely to have diethylphosphate concentrations above the 95th percentile estimate of 15.5 μg/L than adults and senior adults. Conversely, for dimethyldithiophosphate, senior adults were 3.8 times and 1.8 times more likely to be above the 95th percentile than adults and adolescents, respectively, while adults were 2.1 times more likely to be above the 95th percentile than the adolescents. Our data indicate that the most vulnerable segments of our population—children and older adults—have higher exposures to OP pesticides than other population segments. However, according to DAP urinary metabolite data, exposures to OP pesticides have declined during the last six years at both the median and 95th percentile levels.
doi:10.3390/ijerph8083063
PMCID: PMC3166728  PMID: 21909292
NHANES; urine; organophosphorus; pesticide; dialkylphosphate
19.  Analysis of microRNA turnover in mammalian cells following Dicer1 ablation 
Nucleic Acids Research  2011;39(13):5692-5703.
Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function.
doi:10.1093/nar/gkr148
PMCID: PMC3141258  PMID: 21447562
20.  Different modes of interaction by TIAR and HuR with target RNA and DNA 
Nucleic Acids Research  2011;39(3):1117-1130.
TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U–rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2′-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.
doi:10.1093/nar/gkq837
PMCID: PMC3035456  PMID: 21233170
21.  ATF3 transcription factor and its emerging roles in immunity and cancer 
Activating transcription factor 3 (ATF3) is a member of the ATF/cyclic AMP response element-binding (ATF/CREB) family of transcription factors. It is an adaptive-response gene that participates in cellular processes to adapt to extra- and/or intracellular changes, where it transduces signals from various receptors to activate or repress gene expression. Advances made in understanding the immunobiology of Toll-like receptors have recently generated new momentum for the study of ATF3 in immunity. Moreover, the role of ATF3 in the regulation of the cell cycle and apoptosis has important implications for understanding susceptibility to and progression of several cancers.
doi:10.1007/s00109-009-0520-x
PMCID: PMC2783469  PMID: 19705082
ATF3; Transcriptional regulation; Immunity; TLRs; Oncogenesis
22.  Reduced expression of autotaxin predicts survival in uveal melanoma 
The British Journal of Ophthalmology  2007;91(10):1385-1392.
Aim
In an effort to identify patients with uveal melanoma at high risk of metastasis, the authors undertook correlation of gene expression profiles with histopathology data and tumour‐related mortality.
Methods
The RNA was isolated from 27 samples of uveal melanoma from patients who had consented to undergo enucleation, and transcripts profiled using a cDNA array comprised of sequence‐verified cDNA clones representing approximately 4000 genes implicated in cancer development. Two multivariate data mining techniques—hierarchical cluster analysis and multidimensional scaling—were used to investigate the grouping structure in the gene expression data. Cluster analysis was performed with a subset of 10 000 randomly selected genes and the cumulative contribution of all the genes in making the correct grouping was recorded.
Results
Hierarchical cluster analysis and multidimensional scaling revealed two distinct classes. When correlated with the data on metastasis, the two molecular classes corresponded very well to the survival data for the 27 patients. Thirty two discrete genes (corresponding to 44 probe sets) that correctly defined the molecular classes were selected. A single gene (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin) could classify the molecular types. The expression pattern was confirmed using real‐time quantitative PCR.
Conclusions
Gene expression profiling identifies two distinct prognostic classes of uveal melanoma. Underexpression of autotaxin in class 2 uveal melanoma with a poor prognosis needs to be explored further.
doi:10.1136/bjo.2007.116947
PMCID: PMC2001033  PMID: 17475713
23.  Modified vaccinia virus Ankara can activate NF-κB transcription factors through a double-stranded RNA-activated protein kinase (PKR)-dependent pathway during the early phase of virus replication 
Virology  2009;391(2):177-186.
Modified vaccinia virus Ankara (MVA), which is a promising replication-defective vaccine vector, is unusual among the orthopoxviruses in activating NF-κB transcription factors in cells of several types. In human embryonic kidney (HEK 293T) cells, the MVA-induced depletion of IκBα required to activate NF-κB is inhibited by UV-inactivation of the virus, and begins before viral DNA replication. In HEK 293T, CHO, or RK13 cells, expression of the cowpox virus CP77 early gene, or the vaccinia virus K1L early gene suppresses MVA-induced IκBα depletion. In mouse embryonic fibroblasts (MEFs), MVA induction of IκBα depletion is dependent on the expression of mouse or human double-stranded RNA-activated protein kinase (PKR). These results demonstrate that events during the early phase of MVA replication can induce PKR-mediated processes contributing both to the activation of NF-κB signaling, and to processes that may restrict viral replication. This property may contribute to the efficacy of this vaccine virus.
doi:10.1016/j.virol.2009.06.012
PMCID: PMC2765328  PMID: 19596385
vaccinia; MVA; NF-kappaB; I-kappaB; K1L; CP77; cowpox; PKR; orthopoxvirus; vaccine
24.  Role of PKR and Type I IFNs in Viral Control during Primary and Secondary Infection 
PLoS Pathogens  2010;6(6):e1000966.
Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8+ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8+ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8+ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8+ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8+ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8+ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8+ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8+ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines.
Author Summary
Type I interferons (IFNs) constitute the first line of defense against viral infections, promote antigen presentation by dendritic cells, and play a crucial role in directly stimulating anti-viral T cell responses. However, the mechanisms underlying the diverse cellular effects of Type I IFNs are not well defined. One of the best-characterized anti-viral signaling mechanisms induced by Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. We show that requirement for cellular PKR activity could be a distinguishing feature between Type I IFN actions that mediate viral control or stimulate CD8+ T cell expansion during an acute infection with lymphocytic choriomeningitis virus (LCMV). Typically, innate immune mechanisms including Type I IFNs are considered important for viral control during a primary infection. However, we find that presence of vaccine-induced CD8+ T cell memory and accelerated generation of secondary effectors are necessary but not sufficient to provide effective protective immunity to re-infection, without the aid of innate effectors PKR and Type I IFNs. These findings have improved our understanding of virus-immune system interactions and immune correlates of anti-viral protective immunity, which might have implications in the development of effective anti-viral vaccines and immunotherapies.
doi:10.1371/journal.ppat.1000966
PMCID: PMC2891951  PMID: 20585572
25.  Promyelocytic Leukemia Zinc Finger Protein Regulates Interferon-Mediated Innate Immunity 
Immunity  2009;30(6):802-816.
Summary
Interferons (IFNs) direct innate and acquired immune responses and, accordingly, are used therapeutically to treat a number of diseases, yet the diverse effects they elicit are not fully understood. Here we identify the promyelocytic leukemia zinc finger (PLZF) protein as a previously unrecognized component of the IFN response. IFN stimulates an association between PLZF, the promyelocytic leukemia protein and histone deacetylase 1, to induce a decisive subset of IFN-stimulated genes (ISGs). Consequently, PLZF-deficient mice have a specific ISG defect and as a result are more susceptible to viral infection. This susceptibility correlates with a marked decrease in the expression of the key antiviral mediators and an impaired IFN-mediated induction of natural killer cell function. These results provide new insights into the regulatory mechanisms of IFN signaling and the induction of innate antiviral immunity.
doi:10.1016/j.immuni.2009.04.013
PMCID: PMC2711215  PMID: 19523849

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