S-Nitrosylation-Mediated Reduction of CaV1.2 Surface Expression and Open Probability Underlies Attenuated Vasoconstriction Induced by Nitric Oxide.

BACKGROUND: L-type CaV1.2 calcium channel, the primary gateway for Ca2+ influx in smooth muscles, is widely regulated by multiple posttranslational modifications, such as protein kinase-mediated phosphorylation and nitric oxide-induced S-nitrosylation. However, the effect of S-nitrosylation on CaV1.2 channel function and its role in arterial contractility are not well understood. METHODS: Electrophysiological recordings, Ca2+ and confocal imaging, and biochemical assays were used to functionally characterize S-nitrosylated CaV1.2 channels in vitro, while pressure myography and tail-cuff blood pressure measurement were conducted to evaluate the physiological effects of CaV1.2 S-nitrosylation ex vivo and in vivo. RESULTS: S-nitrosylation significantly reduced the CaV1.2 current density by promoting lysosomal degradation that leads to decreased levels of total and surface CaV1.2 channel proteins in a CaVß-independent manner and reducing the open probability of CaV1.2 channel. Mechanistically, the Cys1180 and Cys1280 residues within CaV1.2 channel have been determined as the molecular targets for S-nitrosylation as substitution of either Cys1180 or Cys1280 for serine resulted in substantial reduction of S-nitrosylation levels. Of note, CaV1.2 S-nitrosylation levels were significantly reduced in arteries isolated from both spontaneously hypertensive rats and patients with pulmonary hypertension. Moreover, mouse resistance arteries incubated with S-nitrosocysteine displayed much lower contractility and spontaneously hypertensive rats injected with S-nitrosocysteine also showed significantly reduced blood pressure, suggesting that reduced S-nitrosylation contributes to the upregulation of CaV1.2 channel activity in hypertensive arteries. CONCLUSIONS: This study provides strong evidence that S-nitrosylation-mediated downregulation of CaV1.2 channels is via 2 distinctive mechanisms and the findings offer potential pathways for therapeutic inventions in hypertension.

Performance of the coronary calcium score in an outpatient chest pain clinic and strategies for risk stratification.

BACKGROUND: Coronary artery calcium score (CAC) is an objective marker of atherosclerosis. The primary aim is to assess CAC as a risk classifier in stable coronary artery disease (CAD). HYPOTHESIS: CAC improves CAD risk prediction, compared to conventional risk scoring, even in the absence of cardiovascular risk factor inputs. METHODS: Outpatients presenting to a cardiology clinic (n = 3518) were divided into two cohorts: derivation (n = 2344 patients) and validation (n = 1174 patients). Adding logarithmic transformation of CAC, we built two logistic regression models: Model 1 with chest pain history and risk factors and Model 2 including chest pain history only without risk factors simulating patients with undiagnosed comorbidities. The CAD I Consortium Score (CCS) was the conventional reference risk score used. The primary outcome was the presence of coronary artery disease defined as any epicardial artery stenosis≥50% on CT coronary angiogram. RESULTS: Area under curve (AUC) of CCS in our validation cohort was 0.80. The AUC of Models 1 and 2 were significantly improved at 0.88 (95%CI 0.86-0.91) and 0.87 (95%CI 0.84-0.90), respectively. Integrated discriminant improvement was >15% for both models. At a pre-specified cut-off of ≤10% for excluding coronary artery disease, the sensitivity and specificity were 89.3% and 74.7% for Model 1, and 88.1% and 71.8% for Model 2. CONCLUSION: CAC helps improve risk classification in patients with chest pain, even in the absence of prior risk factor screening.

Immediate effects of valgus bracing on knee joint moments during walking in knee-healthy individuals: Potential modifying effects of body height.

BACKGROUND: The goal of valgus knee brace treatment is to reduce medial knee joint loading during walking, often indicated by external knee adduction moment (KAM) measures. However, existing healthy-subjects studies have been equivocal in demonstrating KAM reduction with valgus knee bracing. RESEARCH QUESTION: What are the immediate effects of valgus bracing at different tension levels on KAM during walking at a controlled speed and does body height modify the brace-KAM associations? METHODS: Data from 32 knee-healthy participants were analysed in this randomized crossover trial. Participants performed walking trials at controlled speed (1.3 ± 0.065 m/s) both with and without an Ossür Unloader One® brace. During the bracing condition, valgus tension was incrementally increased, from zero tension to normal tension and to maximum tolerable tension. RESULTS: Valgus bracing minimally increased knee flexion at heel-strike (P < 0.001) in a dose-dependent manner and minimally reduced gait velocity (∼0.015m/s) across all tension levels. Valgus bracing, overall, did not significantly reduce the various KAM measures. However, brace use at maximal tension was associated with a 0.04Nm/kg (9.2 %) increase in first peak KAM amongst participants with a body height of 1.75 m and a 0.03Nm/kg (7.6 %) decrease in first peak KAM amongst participants with a body height of 1.55 m. SIGNIFICANCE: Valgus bracing did not reduce the various KAM measures during walking; however, body height may play a moderating role. Given knee brace sizes vary more in circumference than length, this result may be due to the ratio between effective moment arm length relative to limb length. A deeper understanding of the potential neuro-biomechanical effects of valgus knee bracing and how these effects are potentially modified by body height may be critical to the design of effective knee braces.

Test-retest reliability and variability of knee adduction moment peak, impulse and loading rate during walking.

BACKGROUND: Previous studies have reported good test-retest reliability for peak knee adduction moment (KAM) during walking. However, reliability of other KAM measurements has not been established. RESEARCH QUESTION: What is the test-retest reliability of peak KAM, KAM impulse, and KAM loading rate measurements during walking in knee-healthy individuals? METHODS: Data from 32 knee-healthy participants were analysed in this test-retest reliability study. Various KAM measurements were reported for two sessions with kinematic and kinetic data obtained from a motion capture system synchronised with force plates, with a median of 1 week between sessions. RESULTS: For all KAM measures, intra-class correlation coefficients were above 0.90 and their lower bound 95 % confidence limits exceeded 0.81. However, absolute measurement variability differed across measures, with normalized SEM (8 %-15 %), normalized MDC95 (20 %-40 %), intra-session MAD (10 %-18 %), and inter-session MAD (12 %-22 %) varying over a 2-fold range. Overall and first peak KAM, KAM impulse over 50 % stance, and KAM loading rate (15 frame window) showed ≤10 % and ≤15 % intra- and inter-session MAD, respectively. SIGNIFICANCE: This study provided previously undefined test-retest reliability estimates for various KAM measures during walking. Researchers and clinicians should not assume that the various aspects of the KAM curve share similar reliability.

Molecular characterization, light-dependent expression, and cellular localization of a host vacuolar-type H+-ATPase (VHA) subunit A in the giant clam, Tridacna squamosa, indicate the involvement of the host VHA in the uptake of inorganic carbon and its supply to the symbiotic zooxanthellae.

The giant clam, Tridacna squamosa, represents a clam-zooxanthellae association. In light, the host clam and the symbiotic zooxanthellae conduct light-enhanced calcification and photosynthesis, respectively. We had cloned the cDNA coding sequence of a Vacuolar-type Proton ATPase (VHA) subunit A, ATP6V1A, from T. squamosa, whereby the VHA is an electrogenic transporter that actively 'pumps' H+ out of the cell. The ATP6V1A of T. squamosa comprised 1866 bp, encoding a protein of 622 amino acids and 69.9 kDa, and had a host-origin. Its gene expression was strong in the ctenidium and the colorful outer mantle, but weak in the whitish inner mantle, corroborating a previous proposition that VHA might have a trivial role in light-enhanced calcification. Light exposure led to significant increases in the gene and protein expression levels of ATP6V1A/ATP6V1A in the ctenidium and the outer mantle. In the ctenidium, the ATP6V1A was localized in the apical epithelia of the filaments and tertiary water channels, indicating that the VHA could participate in the increased excretion of H+ produced during light-enhanced calcification. Additionally, the excreted H+ would augment HCO3- dehydration in the external medium and facilitate the uptake of CO2 by the ctenidium during insolation. In the outer mantle, the ATP6V1A was detected in intracellular vesicles in a type of cells, presumably iridocytes, surrounding the zooxanthellal tubules, and in the apical epithelium of zooxanthellal tubules. Hence, the host VHA could participate in the transfer of inorganic carbon from the hemolymph to the luminal fluid of the tubules by increasing the supply of H+ for the dehydration of HCO3- to CO2 during insolation to benefit the photosynthesizing zooxanthellae.

Whole-genome sequencing analysis of phenotypic heterogeneity and anticipation in Li-Fraumeni cancer predisposition syndrome.

The Li-Fraumeni syndrome (LFS) and its variant form (LFL) is a familial predisposition to multiple forms of childhood, adolescent, and adult cancers associated with germ-line mutation in the TP53 tumor suppressor gene. Individual disparities in tumor patterns are compounded by acceleration of cancer onset with successive generations. It has been suggested that this apparent anticipation pattern may result from germ-line genomic instability in TP53 mutation carriers, causing increased DNA copy-number variations (CNVs) with successive generations. To address the genetic basis of phenotypic disparities of LFS/LFL, we performed whole-genome sequencing (WGS) of 13 subjects from two generations of an LFS kindred. Neither de novo CNV nor significant difference in total CNV was detected in relation with successive generations or with age at cancer onset. These observations were consistent with an experimental mouse model system showing that trp53 deficiency in the germ line of father or mother did not increase CNV occurrence in the offspring. On the other hand, individual records on 1,771 TP53 mutation carriers from 294 pedigrees were compiled to assess genetic anticipation patterns (International Agency for Research on Cancer TP53 database). No strictly defined anticipation pattern was observed. Rather, in multigeneration families, cancer onset was delayed in older compared with recent generations. These observations support an alternative model for apparent anticipation in which rare variants from noncarrier parents may attenuate constitutive resistance to tumorigenesis in the offspring of TP53 mutation carriers with late cancer onset.

Cholangitis in children with biliary atresia: health-care resource utilisation.

AIM: Cholangitis is a well-known complication that contributes to morbidity, mortality, as well as health-care utilisation in children with biliary atresia who have undergone the Kasai portoenterostomy. The aim of the study was to determine the common causative organisms for cholangitis and characterise its burden, health-care resource and service utilisation and cost. METHODS: This was a retrospective chart review of children who underwent Kasai portoenterostomy in our institution from 1988 to 2011. The causative organisms were identified based on culture reports. The burden of the disease was estimated based on the number of patients experiencing one or more episodes of cholangitis. Health-care resource and service utilisation were based on different categories, and cost was computed based on the charges at the institution. RESULTS: Twenty-seven (64.3%) out of 42 children included in the analysis experienced at least one episode of cholangitis. There were a total of 97 episodes of cholangitis, with an average of 3.6 (1-15) episodes per patient. The average length of stay per episode of cholangitis was 14.8 (2-64) days. Common organisms isolated during blood cultures were Klebsiella pneumoniae, Enterococcus, Escherichia coli and Pseudomonas aeruginosa. The estimated cost per in-patient admission of 15 days (rounded off) for a single episode of cholangitis was $SG 8986.61 ($US 7369.02). CONCLUSION: The knowledge about the incidence and cost of cholangitis will allow physicians to counsel parents of children newly diagnosed with biliary atresia and to better prepare them both emotionally and financially for what to expect.

Prevalence of cognitive impairment in Chinese: epidemiology of dementia in Singapore study.

OBJECTIVE: To study the prevalence of and associated factors for cognitive impairment and dementia in community dwelling Chinese from Singapore. METHODS: This study includes Chinese subjects from the Epidemiology of Dementia in Singapore (EDIS) study, aged ≥60 years, who underwent comprehensive examinations, including cognitive screening with the locally validated Abbreviated Mental Test and Progressive Forgetfulness Questionnaire. Screen positive participants subsequently underwent extensive neuropsychological testing and cerebral MRI. Cognitive impairment no dementia (CIND) and dementia were diagnosed according to internationally accepted criteria. The prevalence of cognitive impairment and dementia were computed per 5 year age categories and gender. To examine the relationship between baseline associated factors and cognitive impairment, we used logistic regression models to compute odd ratios with 95% CI. RESULTS: 1538 Chinese subjects, aged ≥60 years, underwent cognitive screening: 171 (15.2%) were diagnosed with any cognitive impairment, of whom 84 were CIND mild, 80 CIND moderate and seven had dementia. The overall age adjusted prevalence of CIND mild was 7.2%; CIND moderate/dementia was 7.9%. The prevalence increased with age, from 5.9% in those aged 60-64 years to 31.3% in those aged 75-79 years and 44.1% in those aged ≥80 years. Multivariate analysis revealed age, diabetes and hyperlipidaemia to be independently associated with cognitive impairment. CONCLUSIONS: In present study, the overall prevalence of cognitive impairment and dementia in Chinese was 15.2%, which is in the same range as the prevalence reported in Caucasian and other Asian populations.

The informant AD8 is superior to participant AD8 in detecting cognitive impairment in a memory clinic setting.

The informant AD8 has good discriminatory indices in detecting questionable dementia. However, studies on participant AD8 yielded inconsistent results. This study aims to investigate the discriminatory ability of the AD8 in detecting cognitive impairment at a memory clinic by comparing the informant AD8, participant AD8, Montreal Cognitive Assessment (MoCA), and Mini-Mental State Examination (MMSE). The AD8 was administered to 280 participant-informant dyads. The MoCA and MMSE were administered to all participants, who subsequently received a comprehensive clinical and neuropsychological assessment leading to a consensus diagnosis and a Clinical Dementia Rating (CDR). Area under the receiver operating characteristic curve (ROC) analysis was used to compare the discriminatory ability of AD8, MoCA, and MMSE. Participants were Chinese (83.6%) females (54.3%) with a mean age and education of 73.4 ± 8.6 years and 6.2 ± 5.6 years, respectively. The discriminant validity of the informant AD8 was significantly superior to the participant AD8 in detecting cognitive impairment (CDR ≥ 0.5) {Area Under Curve (AUC) [95% confidence interval (CI)]: 0.96 (0.93-0.98) versus 0.66 (0.58-0.74), p < 0.01}. Furthermore, the informant AD8 was equivalent to MoCA and MMSE in detecting cognitive impairment {AUC [95% CI]: MoCA [0.98 (0.96-0.99)]; MMSE [0.95 (0.93-0.98)]}. The informant AD8 (≥2) had very good sensitivity and specificity, while the participant AD8 (≥2) had suboptimal sensitivity and specificity in detecting cognitive impairment (sensitivity 0.93 versus 0.59; specificity 0.87 versus 0.65; 91.8% versus 60% correctly classified). The informant AD8 is superior to the participant AD8, and equivalent to the MMSE and MoCA in screening for cognitive impairment in memory clinic patients.