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Background Diabetes foot ulcer (DFU) is a complication of diabetes mellitus. Accurate diagnosis of DFU severity through inflammatory markers will assist in reducing impact on quality of life. We aimed to ascertain the diagnostic test accuracy of commonly used inflammatory markers such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), procalcitonin (PCT), and white cell count (WCC) for the diagnosis and differentiation between DFU grades based on the International Working Group on the Diabetic Foot classification system. Methods This systematic review explored studies that investigated one or more of the above-listed index tests aiding in diagnosing infected DFU. This review was registered on PROSPERO database (ID = CRD42021255618) and searched 5 databases including an assessment of the references of included studies. Records were manually screened as per Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. A total of 16 studies were included which were assessed for quality using QUADAS-2 tool and meta-analysed using Meta-Disc v1.4. Results CRP had the greatest area under the curve (AUC) of 0.893 for diagnosing grade 2 DFU. This returned a pooled sensitivity and specificity of 77.4% (95% CI: 72% to 82%) and 84.3% (95% CI: 79% to 89%) respectively. In terms of diagnosing grade 3 DFU, procalcitonin had the highest AUC value of 0.844 when compared with other markers. The pooled sensitivity of PCT was calculated as 85.5% (95% CI: 79% to 90%) and specificity as 68.9% (95% CI: 63% to 75%). Conclusion CRP and PCT are the best markers for diagnosing grade 2 and grade 3 DFU respectively. Other markers are also valuable when used in conjunction with clinical judgement. The findings accentuate the necessity of further research to establish standardised cut-off values for these inflammatory markers in diagnosing diabetic foot ulcers.

Otosclerosis (OTSC) is the primary form of conductive hearing loss characterized by abnormal bone remodelling within the otic capsule of the human middle ear. A genetic association of the RELN SNP rs3914132 with OTSC has been identified in European population. Previously, we showed a trend towards association of this polymorphism with OTSC and identified a rare variant rs74503667 in a familial case. Here, we genotyped these variants in an Indian cohort composed of 254 OTSC cases and 262 controls. We detected a significant association of rs3914132 with OTSC (OR = 0.569, 95%CI = 0.386–0.838, p = 0.0041). To confirm this finding, we completed a meta-analysis which revealed a significant association of the rs3914132 polymorphism with OTSC (Z = 6.707, pRELN locus (LOD score 2.1059) in the OTSC family which has shown the transmission of rare variant rs74503667 in the affected individuals. To understand the role of RELN and its receptors in the development of OTSC, we went further to perform a functional analysis of RELN/reelin. Here we detected a reduced RELN (p = 0.0068) and VLDLR (p = 0.0348) mRNA levels in the otosclerotic stapes tissues. Furthermore, a reduced reelin protein expression by immunohistochemistry was confirmed in the otosclerotic tissues. Electrophoretic mobility shift assays for rs3914132 and rs74503667 variants revealed an altered binding of transcription factors in the mutated sequences which indicates the regulatory role of these variations in the RELN gene regulation. Subsequently, we showed by scanning electron microscopy a change in stapes bone morphology of otosclerotic patients. In conclusion, this study evidenced that the rare variation rs74503667 and the common polymorphism rs3914132 in the RELN gene and its reduced expressions that were associated with OTSC.

As blood-derived miRNAs (c-miRNAs) are modulated by exercise and nutrition, we postulated that they might be used to monitor the effects of a lifestyle intervention (LI) to prevent diabetes development. To challenge this hypothesis, obese Asian Indian pre-diabetic patients were submitted to diet modifications and physical activity for 4 months (LI group) and compared to a control group which was given recommendations only. We have considered 2 periods of time to analyze the data, i.e.; a first one to study the response to the intervention (4 months), and a second one post-intervention (8 months). At basal, 4 months and 8 months post-intervention the levels of 17 c-miRNAs were quantified, selected either for their relevance to the pathology or because they are known to be modulated by physical activity or diet. Their variations were correlated with variations of 25 metabolic and anthropometric parameters and cytokines. As expected, fasting-glycaemia, insulin-sensitivity, levels of exercise- and obesity-induced cytokines were ameliorated after 4 months. In addition, the levels of 4 miRNAs (i.e.; miR-128-3p, miR-374a-5p, miR-221-3p, and miR-133a-3p) were changed only in the LI group and were correlated with metabolic improvement (insulin sensitivity, cytokine levels, waist circumference and systolic blood pressure). However, 8 months post-intervention almost all ameliorated metabolic parameters declined indicating that the volunteers did not continue the protocol on their own. Surprisingly, the LI positive effects on c-miRNA levels were still detected, and were even more pronounced 8 months post-intervention. In parallel, MCP-1, involved in tissue infiltration by immune cells, and Il-6, adiponectin and irisin, which have anti-inflammatory effects, continued to be significantly and positively modified, 8 months post-intervention. These data demonstrated for the first time, that c-miRNA correlations with metabolic parameters and insulin sensitivity are in fact only indirect and likely associated with the level systemic inflammation. More generally speaking, this important result explains the high variability between the previous studies designed to identify specific c-miRNAs associated with the severity of insulin-resistance. The results of all these studies should take into account the level of inflammation of the patients. In addition, this finding could also explain why, whatever the pathology considered (i.e.; cancers, diabetes, neurodegenerative disorders, inflammatory diseases) the same subset of miRNAs is always found altered in the blood of patients vs healthy subjects, as these pathologies are all associated with the development of inflammation.

Background Nearly 1/3rd of patients undergoing coronary artery bypass graft surgery (CABG) have left ventricular systolic dysfunction. However, the extent, direction and implications of perioperative changes in left ventricular ejection fraction (LVEF) have not been well characterized in these patients. Methods We studied the changes in LVEF among 549 patients with left ventricular systolic dysfunction (LVEF 10% in LVEF was considered clinically significant. Results Of the 549 patients (mean age 61.4±9.55 years, and 72 [13.1%] women), 145 (26.4%) had a >10% improvement in LVEF, 369 (67.2%) had no change and 35 (6.4%) had >10% worsening of LVEF following CABG. Patients with lower preoperative LVEF were more likely to experience an improvement after CABG (odds ratio 1.36; 95% CI 1.21–1.53; per 5% lower preoperative LVEF; p 10% improvement in LVEF after CABG was associated with a 57% lower risk of all-cause mortality (HR: 0.43, 95% CI: 0.26–0.71). Conclusions Among patients with ischemic cardiomyopathy undergoing CABG, 26.4% had >10% improvement in LVEF. An improvement in LVEF was more likely in patients with lower preoperative LVEF and was associated with improved long-term survival.

IntroductionCreating a proper career program is the best way to enhance employees’ organizational commitment; it motivates and retains them. This research aims to measure career growth’s influence on turnover intention, mediated by employees’ commitment through self-reported employees’ perceptions. This study identifies the key dimensions of organizational commitment (affective, continuance and normative commitment) that mediate the relationship between career growth and employee turnover intention exploring the indirect effects between career growth and turnover intention. The relationship is examined among the public sector employees in the Sultanate of Oman, a sector currently facing high turnover rates and losing key skilled talent pool.MethodologyData collection was executed through an adopted questionnaire distributed among 500 employees of 38 government units within the Sultanate of Oman. A total of 351 questionnaires were returned, and after the initial screening process, 329 were found to be valid for further analysis. CFA analysis was conducted to identify the factors falling under the three primary constructs of the study. Assessment of the models was explained through Goodness-of-fit Indices. Structural equation modeling, which is most recommended to study the effect of latent variables, was performed using AMOS to evaluate the mediating role of organizational commitment between career growth and employee turnover intention.FindingsThe results indicated that the potential for career growth is an essential motivating element for public sector employees in the Sultanate of Oman to encourage retention and reduce intention to leave. The findings also confirm that effective and continuous commitment significantly mediates the relationship between career growth and employees’ turnover intention.Contribution and originality valueThe results generated could help both researchers and those involved in public sector policy-making to understand how employee turnover intention is influenced by career growth and organizational commitment in the public sector in the Sultanate of Oman.

Motivation Annotation of large amounts of generated sequencing data is a demanding task. Most of the currently available robust annotation tools, like ANNOVAR, are command-line based tools which require a certain degree of programming skills. User-friendly tools for variant annotation of sequencing data with graphical interface are under-represented. Results We have developed an interactive application, which harnesses the easy usability of R Shiny and combines it with the versatile annotation features of ANNOVAR. This application is easy to use and gives comprehensive annotations for user supplied vcf files using multiples databases. The output table contains the list of variants and their corresponding annotation presented within the graphical interface. In addition, the annotation results are downloadable as text file.

Low-end LiDAR sensor provides an alternative for depth measurement and object recognition for lightweight devices. However due to low computing capacity, complicated algorithms are incompatible to be performed on the device, with sparse information further limits the feature available for extraction. Therefore, a classification method which could receive sparse input, while providing ample leverage for the classification process to accurately differentiate objects within limited computing capability is required. To achieve reliable feature extraction from a sparse LiDAR point cloud, this paper proposes a novel Clustered Extraction and Centroid Based Clustered Extraction Method (CE-CBCE) method for feature extraction followed by a convolutional neural network (CNN) object classifier. The integration of the CE-CBCE and CNN methods enable us to utilize lightweight actuated LiDAR input and provides low computing means of classification while maintaining accurate detection. Based on genuine LiDAR data, the final result shows reliable accuracy of 97% through the method proposed.

Background With increasing use of nCPAP, the safety and comfort associated with nCPAP have come into the forefront. The reported incidence of nasal injuries associated with the use of nCPAP is 20% to 60%. A recent meta-analysis concluded that the use of nasal masks significantly decreases CPAP failure and the incidence of moderate to severe nasal injury and stress the need for a well powered RCT to confirm their findings. Methods In this Open label, 3 arms, sequential, stratified randomized controlled trial, we evaluated the incidence and severity of nasal injury at removal of nCPAP when using two different nasal interfaces and in three groups (i.e. rotation group, mask continue group, prong continue group). Preterm infants with gestation ≤ 30 weeks and respiratory distress within the first 6 hours of birth and in need of CPAP were eligible for the study. Results Among the 175 newborns included in the study, incidence of nasal injury in mask continue group [n = 19/57 (33.3%)] was significantly less as compared to prong continue group [n = 55/60 (91.6%)] and rotation group [33/ 58 (56.9%), p value

The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to ≤ 10 μg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intra-laboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSDR) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The excellent performance of the xMAP FADA when performed by analysts of varying proficiency indicates a reliability sufficient to meet analytical needs.

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70-80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

BACKGROUND: We comparatively assessed the performance of six simple obesity indices to identify adults with cardiovascular disease (CVD) risk factors in a diverse and contemporary South Asian population. METHODS: 8,892 participants aged 20-60 years in 2010-2011 were analyzed. Six obesity indices were examined: body mass index (BMI), waist circumference (WC), waist-height ratio (WHtR), waist-hip ratio (WHR), log of the sum of triceps and subscapular skin fold thickness (LTS), and percent body fat derived from bioelectric impedance analysis (BIA). We estimated models with obesity indices specified as deciles and as continuous linear variables to predict prevalent hypertension, diabetes, and high cholesterol and report associations (prevalence ratios, PRs), discrimination (area-under-the-curve, AUCs), and calibration (index χ2). We also examined a composite unhealthy cardiovascular profile score summarizing glucose, lipids, and blood pressure. RESULTS: No single obesity index consistently performed statistically significantly better than the others across the outcome models. Based on point estimates, WHtR trended towards best performance in classifying diabetes (PR = 1.58 [1.45-1.72], AUC = 0.77, men; PR = 1.59 [1.47-1.71], AUC = 0.80, women) and hypertension (PR = 1.34 [1.26,1.42], AUC = 0.70, men; PR = 1.41 [1.33,1.50], AUC = 0.78, women). WC (mean difference = 0.24 SD [0.21-0.27]) and WHtR (mean difference = 0.24 SD [0.21,0.28]) had the strongest associations with the composite unhealthy cardiovascular profile score in women but not in men. CONCLUSIONS: WC and WHtR were the most useful indices for identifying South Asian adults with prevalent diabetes and hypertension. Collection of waist circumference data in South Asian health surveys will be informative for population-based CVD surveillance efforts.

BACKGROUND Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) are far more prevalent in European Americans than in African Americans. Hypothesizing that this racial disparity in prevalence might represent a genetic susceptibility, we used an admixture mapping approach to interrogate disease association with genomic differences between European and African ancestry. METHODS Formalin fixed paraffin embedded samples were identified from 54 African Americans with BE or EAC through review of surgical pathology databases at participating Barrett's Esophagus Translational Research Network (BETRNet) institutions. DNA was extracted from normal tissue, and genotyped on the Illumina OmniQuad SNP chip. Case-only admixture mapping analysis was performed on the data from both all 54 cases and also on a subset of 28 cases with high genotyping quality. Haplotype phases were inferred with Beagle 3.3.2, and local African and European ancestries were inferred with SABER plus. Disease association was tested by estimating and testing excess European ancestry and contrasting it to excess African ancestry. RESULTS Both datasets, the 54 cases and the 28 cases, identified two admixture regions. An association of excess European ancestry on chromosome 11p reached a 5% genome-wide significance threshold, corresponding to -log10(P) = 4.28. A second peak on chromosome 8q reached -log10(P) = 2.73. The converse analysis examining excess African ancestry found no genetic regions with significant excess African ancestry associated with BE and EAC. On average, the regions on chromosomes 8q and 11p showed excess European ancestry of 15% and 20%, respectively. CONCLUSIONS Chromosomal regions on 11p15 and 8q22-24 are associated with excess European ancestry in African Americans with BE and EAC. Because GWAS have not reported any variants in these two regions, low frequency and/or rare disease associated variants that confer susceptibility to developing BE and EAC may be driving the observed European ancestry association evidence.

Multiple statistical approaches have been proposed to validate reference genes in qPCR assays. However, conflicting results from these statistical methods pose a major hurdle in the choice of the best reference genes. Recent studies have proposed the use of a minimum of three different methods but there is no consensus on how to interpret conflicting results. Researchers resort to averaging the ranks or attributing a weighted rank to candidate genes. However, we report here that the suitability of these validation methods can be influenced by the experimental setting. Therefore, averaging the ranks can lead to suboptimal assessment of stable reference genes if the method used is not suitable for analysis. As the respective approaches of these statistical methods are different, a clear understanding of the fundamental assumptions and the parameters that influence the reference gene stability calculation is necessary. In this study, the stability of 10 candidate reference genes (Actb, Gapdh, Tbp, Sdha, Pgk1, Ppia, Rpl13a, Hsp60, Mrpl10, Rps26) was assessed using four common statistical approaches (GeNorm, NormFinder, Coefficient of Variation or CV analysis and Pairwise ΔCt method) in a longitudinal experimental setting. We used the development of the cerebellum and the spinal cord of mice as a model to assess the suitability of these statistical methods for reference gene validation. GeNorm and the Pairwise ΔCt were found to be ill suited due to a fundamental assumption in their stability calculations. Highly correlated genes were given better stability ranks despite significant overall variation. NormFinder fares better but the presence of highly variable genes influences the ranking of all genes because of the algorithm’s construct. CV analysis estimates overall variation, but it fails to consider variation across groups. We thus highlight the assumptions and potential pit-falls of each method using our longitudinal data. Based on our results, we have devised a workflow combining NormFinder, CV analysis along with visual representation of mRNA fold changes and one-way ANOVA for validating reference genes in longitudinal studies. This workflow proves to be more robust than any of these methods used individually. Additional Information Competing Interests – The authors declare no conflict of interest.

This open-label study assessed the safety and immunogenicity of two doses (14 days apart) of an indigenously manufactured, killed, bivalent (Vibrio cholerae O1 and O139), whole-cell oral cholera vaccine (SHANCHOL; Shantha Biotechnics) in healthy adults (n = 100) and children (n = 100) in a cholera endemic area (Vellore, South India) to fulfill post-licensure regulatory requirements and post-World Health Organization (WHO) prequalification commitments. Safety and reactogenicity were assessed, and seroconversion rates (i.e. proportion of participants with a ≥ 4-fold rise from baseline in serum vibriocidal antibody titers against V. cholerae O1 Inaba, O1 Ogawa and O139, respectively) were determined 14 days after each vaccine dose. No serious adverse events were reported during the study. Commonly reported solicited adverse events were headache and general ill feeling. Seroconversion rates after the first and second dose in adults were 67.7% and 55.2%, respectively, against O1 Inaba; 47.9% and 45.8% against O1 Ogawa; and 19.8% and 20.8% against O139. In children, seroconversion rates after the first and second dose were 80.2% and 68.8%, respectively, against O1 Inaba; 72.9% and 67.7% against O1 Ogawa; and 26.0% and 18.8% against O139. The geometric mean titers against O1 Inaba, O1 Ogawa, and O139 in both adults and children were significantly higher after each vaccine dose compared to baseline titers (P < 0.001; for both age groups after each dose versus baseline). The seroconversion rates for O1 Inaba, O1 Ogawa, and O139 in both age groups were similar to those in previous studies with the vaccine. In conclusion, the killed, bivalent, whole-cell oral cholera vaccine has a good safety and reactogenicity profile, and is immunogenic in healthy adults and children. Trial Registration: ClinicalTrials.gov NCT00760825; CTRI/2012/01/002354.

Background and Aim Because ethanol consumption is commonly associated with a high cholesterol diet, we examined whether combined consumption of ethanol and high cholesterol increases liver injury and fibrosis. Methods Male C57BL/6J mice were fed diets containing: 1) 35% of calories from corn oil (CTR), 2) CTR plus 0.5% (w/v) cholesterol (Chol), 3) CTR plus ethanol (27% of calories) (EtOH), or 4) EtOH+Chol for 3 months. Results In mice fed Chol or EtOH alone, ALT increased to ~160 U/L, moderate hepatic steatosis occurred, and leukocyte infiltration, necrosis, and apoptosis increased modestly, but no observable fibrosis developed. By contrast in mice fed EtOH+Chol, ALT increased to ~270 U/L, steatosis was more extensive and mostly macrovesicular, and expression of proinflammatory molecules (HMGB-1, TLR4, TNFα, ICAM-1) and leukocyte infiltration increased substantially. Necrosis and apoptosis also increased. Trichrome staining and second harmonic generation microscopy revealed hepatic fibrosis. Fibrosis was mostly sinusoidal and/or perivenular, but in some mice bridging fibrosis occurred. Expression of smooth muscle α-actin and TGF-β1 increased slightly by Chol, moderately by EtOH, and markedly by EtOH+Chol. TGF-β pseudoreceptor BAMBI increased slightly by Chol, remained unchanged by EtOH and decreased by EtOH+Chol. MicroRNA-33a, which enhances TGF-β fibrotic effects, and phospho-Smad2/3, the down-stream signal of TGF-β, also increased more greatly by EtOH+Chol than Chol or EtOH. Metalloproteinase-2 and -9 were decreased only by EtOH+Chol. Conclusion High dietary cholesterol and chronic ethanol consumption synergistically increase liver injury, inflammation, and profibrotic responses and suppress antifibrotic responses, leading to severe steatohepatitis and early fibrosis in mice.

Objective To investigate associations between retinal microvascular changes and cognitive impairment in newly diagnosed type 2 diabetes mellitus. Design Case control study. Setting A primary care cohort with newly diagnosed type 2 diabetes mellitus. Methods For this analysis, we compared 69 cases with lowest decile scores (for the cohort) on the Modified Telephone Interview for Cognitive Status and 68 controls randomly selected from the remainder of the cohort. Retinal images were rated and the following measures compared between cases and controls: retinal vessel calibre, arterio-venous ratio, retinal fractal dimension, and simple and curvature retinal vessel tortuosity. Results Total and venular (but not arteriolar) simple retinal vessel tortuosity levels were significantly higher in cases than controls (t = 2.45, p = 0.015; t = 2.53, p = 0.013 respectively). The associations persisted after adjustment for demographic factors, retinopathy, neuropathy, obesity and blood pressure. There were no other significant differences between cases and controls in retinal measures. Conclusions A novel association was found between higher venular tortuosity and cognitive impairment in newly diagnosed type 2 diabetes mellitus. This might be accounted for by factors such as hypoxia, thrombus formation, increased vasoendothelial growth factor release and inflammation affecting both the visible retinal and the unobserved cerebral microvasculature.

Despite availability of high quality medical records, health care systems often do not have the resources or tools to utilize these data efficiently. Yet, hospital-based, laboratory-confirmed records may pave the way for building reliable surveillance systems capable of monitoring temporal trends of emerging infections. In this communication, we present a new tool to compress and visualize medical records with a local population profile (LPP) approach, which transforms information into statistically comparable patterns. We provide a step-by-step tutorial on how to build, interpret, and expand the use of LPP using hospitalization records of laboratory-confirmed cholera. We abstracted case information from the databases maintained by the Department of Clinical Microbiology at Christian Medical College in Vellore, India. We used a single-year age distribution to construct LPPs for O1, O139, and non O1/O139 serotypes of Vibrio cholerae. Disease counts and hospitalization rates were converted into fitted kernel-based probability densities. We formally compared LPPs with the Kolmogorov-Smirnov test, and created multi-panel visuals to depict temporal trend, age distribution, and hospitalization rates simultaneously. Our first implementation of LPPs revealed information that is typically gathered from surveillance systems such as: i) estimates of the demographic distribution of diseases and identification of a population at risk, ii) changes in the dominant pathogen presence; and iii) trends in disease occurrence. The LPP demonstrated the benefit of increased resolution in pattern detection of disease for different Vibrio cholerae serotypes and two demographic categories by showing patterns and anomalies that would be obscured by traditional methods of analysis and visualization. LPP can be used effectively to compile basic patient information such as age, sex, diagnosis, location, and time into compact visuals. Future development of the proposed approach will allow public health researchers and practitioners to broadly utilize and efficiently compress large volumes of medical records without loss of information.

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1 in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether, our results show that knockdown of CDK2AP1 in primary human fibroblasts reduced proliferation and induced premature senescence, with the observed phenotype being p53 dependent.

Objective We investigated the role of state-level differences in modifiable cardiovascular (CV) risk factors in contributing to state disparities in cardiovascular mortality rates in the US. Methods Adults aged 45–74 in 2010 were examined. We constructed a CV risk index summarizing state-level exposure to current smoking, obesity, physical inactivity, alcohol abstinence, hypertension, elevated cholesterol, and diabetes using the Behavioral Risk Factor Surveillance System. Outcomes were cardiovascular, coronary heart disease, and stroke mortality. Linear regression was used to estimate associations between the CV risk index and mortality outcomes. Models accounted for state-level socioeconomic characteristics and other potential confounders. Results Risk factors were highly correlated at the state-level (Cronbach's alpha 0.85 (men) and 0.92 (women). Each +1SD difference in the cardiovascular risk index was associated with higher adjusted cardiovascular mortality rates by 41.0 (95%CI = 26.3, 55.7) and 33.3 (95%CI = 24.4, 42.2) deaths per 100,000 for men and women, respectively. The index accounted for 8% (men) and 11% (women) of the variation in state-level cardiovascular mortality. Comparable associations were also observed for coronary heart disease and stroke mortality. Conclusions CV risk factors were highly correlated at the state-level and were independently associated with state CV mortality, suggesting the utility of generalized CV risk reduction.

Background/Aims An increase of ethanol metabolism and hepatic mitochondrial respiration occurs in vivo after a single binge of alcohol. Here, our aim was to determine how ethanol intake affects hepatic mitochondrial polarization status in vivo in relation to ethanol metabolism and steatosis. Methods Hepatic mitochondrial polarization, permeability transition (MPT), and reduce pyridine nucleotides, and steatosis in mice were monitored by intravital confocal/multiphoton microscopy of the fluorescence of rhodamine 123 (Rh123), calcein, NAD(P)H, and BODIPY493/503, respectively, after gavage with ethanol (1–6 g/kg). Results Mitochondria depolarized in an all-or-nothing fashion in individual hepatocytes as early as 1 h after alcohol. Depolarization was dose- and time-dependent, peaked after 6 to 12 h and maximally affected 94% of hepatocytes. This mitochondrial depolarization was not due to onset of the MPT. After 24 h, mitochondria of most hepatocytes recovered normal polarization and were indistinguishable from untreated after 7 days. Cell death monitored by propidium iodide staining, histology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was low throughout. After alcohol, mitochondrial NAD(P)H autofluorescence increased and decreased, respectively, in hepatocytes with polarized and depolarized mitochondria. Ethanol also caused steatosis mainly in hepatocytes with depolarized mitochondria. Depolarization was linked to ethanol metabolism, since deficiency of alcohol dehydrogenase and cytochrome-P450 2E1 (CYP2E1), the major ethanol-metabolizing enzymes, decreased mitochondrial depolarization by ~70% and ~20%, respectively. Activation of aldehyde dehydrogenase decreased depolarization, whereas inhibition of aldehyde dehydrogenase enhanced depolarization. Activation of aldehyde dehydrogenase also markedly decreased steatosis. Conclusions Acute ethanol causes reversible hepatic mitochondrial depolarization in vivo that may contribute to steatosis and increased mitochondrial respiration. Onset of this mitochondrial depolarization is linked, at least in part, to metabolism of ethanol to acetaldehyde.