The cellular protein MCM3AP is required for inhibition of cellular DNA synthesis by the IE86 protein of human cytomegalovirus.

Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G(0)-G(1) phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein--an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner

Prospective study of biliary strictures to determine the predictors of malignancy

ORIGINAL ARTICLE BACKGROUND: There have been few prospective studies regarding the investigation of biliary strictures, principally because of rapid technological change. The present study was designed to determine the sensitivity of various imaging studies for the detection of biliary strictures. Serum biochemistry and imaging studies were evaluated for their role in distinguishing benign from malignant strictures. METHODS: Thirty-one patients with suspected noncalculus biliary obstruction were enrolled consecutively in the study. A complete biochemical profile, ultrasound, Disida scan and cholangiogram (endoscopic retrograde cholangiopancreatography [ERCP] or percutaneous cholangiogram) were obtained at study entry. Stricture etiology was determined based on cytology, biopsy and/or clinical follow-up at one year. RESULTS: Twenty-nine of 31 patients had biliary strictures, of which 15 were malignant. The mean age of the malignant cohort was 73.9 years versus 53.9 years in the benign cohort (P<0.001). Statistically significant differences between the malignant and benign groups, respectively, were as follows: alanine transaminase 235.2 versus 66.9 U/L (P=0.004), aspartate transaminase 189.8 versus 84.5 U/L (P=0.011), alkaline phosphatase 840.2 versus 361.1 U/L (P=0.002), bilirubin 317.8 versus 22.1 µmol/L (P<0.001) and bile acids 242.5 versus 73.2 µmol/L (P=0.001). Threshold analysis using receiver operative characteristic (ROC) curves demonstrated that a bilirubin level of 75 µmol/L was most predictive of malignant strictures. Intrahepatic duct dilation was present in 93% of malignant strictures versus 36% of benign strictures (P=0.002). Common hepatic duct dilation was less discriminatory (malignant 13.5 versus benign 9.6 mm; P=0.11). Ultrasound was highly sensitive (93%) in the detection of the primary tumour in the bile duct or pancreas, or in the visualization of nodal or liver metastases. In benign disease, ultrasound failed to detect evidence of intrahepatic or extrahepatic biliary dilation in most cases. Disida scans were not able to distinguish between malignant or benign strictures and could not accurately localize the level of obstruction. The sensitivity of Disida scan for the diagnosis of obstruction was 50%. Cholangiographic characterization of strictures revealed an equal distribution of smooth (eight of 13) and irregular (five of 13) strictures in the malignant group. Ten of 13 benign strictures were characterized as smooth. Malignant strictures were significantly longer than benign ones -30.3 versus 9.2 mm (P=0.001). Threshold analysis using ROC curves showed that strictures greater than or equal to 14 mm were predictive of malignancy (sensitivity 78%, specificity 75%, log odds ratio 11.23). CONCLUSIONS: A serum bilirubin level of 75 µmol/L or higher, or a stricture length of greater than 14 mm was highly predictive of malignancy in patients with a biliary stricture. Ultrasound was useful in predicting malignant strictures by detecting either intrahepatic duct dilation or by visualizing the tumour (primary or metastases). Strictures with a 'benign' cholangiographic appearance are frequently malignant. Disida scan did not add additional information. ERCP is necessary to diagnose benign strictures, which tend to be less extensive at presentation. B iliary strictures are a challenging problem for the clinician. By the time that patients with biliary strictures are referred to a specialist, the diagnosis is usually already known or strongly suspected because clinical evaluation and noninvasive investigations alone have a high specificity and sensitivity (1-4). The job of the medical or surgical specialist is not only to confirm the diagnosis of biliary stricture but also, importantly, to define the etiology and the exact anatomic location, which is vital to therapeutic planning. The differentiation between benign and malignant strictures can be difficult but is of obvious importance in regard to prognosis and optimal therapy. Numerous imaging modalities are available for the investigation of biliary strictures, including abdominal ultrasound, computed tomographic (CT) scanning, nuclear imaging, percutaneous transhepatic cholangiography (PTC), endoscopic retrograde cholangiopancreatography (ERCP) and most recently magnetic resonance cholangiopancreatography (MRCP). Comparative and descriptive studies in this area are lacking, primarily because rapid technological improvements and developments outdate them. We, therefore, embarked on a prospective descriptive trial with the following aims: · Determine the predictive value of liver enzymes, serum bilirubin, serum bile acids, ultrasound and diethyl-iminodiacetic acid (Disida) nuclear imaging for the presence of malignant biliary strictures. · Measure the ability of ultrasound and nuclear imaging to localize the level of obstruction using direct cholangiography as the gold standard. · Determine the sensitivity of abdominal ultrasound and Disida nuclear scanning for the detection of biliary strictures. · Investigate the utility of various cholangiographic features to distinguish malignant from benign strictures. PATIENTS AND METHODS Patients: All patients with biliary strictures referred to the Division of Gastroenterology at the University of Alberta Hospitals for investigation between January 1, 1995 and December 31, 1995 were prospectively entered into the trial. The inclusion criteria were age 16 years or older and noncalculus biliary obstruction. Patients were excluded if subsequent evaluation did not show a stricture. Ethics committee approval was obtained. sente dans 93 % des sténoses malignes par rapport à 36 % des sténoses béni-gnes (P = 0,002). Une dilatation du canal hépatique commun était moins discriminatoire (13,5 mm en cas de malignité par rapport à 9,6 mm en cas de bénignité, P = 0,11). L'échographie était hautement sensible (93 %) pour déceler la tumeur primaire dans le canal biliaire ou le pancréas, ou pour visualiser les nodules ou les métastases hépatiques. Dans le cas d'une maladie bénigne, l'échogra-phie n'a pas réussi à déceler la présence d'une dilatation biliaire intra-hépa-tique ou extra-hépatique dans la plupart des cas. Les scintigraphies au disida n'ont pas pu différencier les sténoses malignes des sténoses bénignes ou localiser précisément le niveau de l'obstruction. La sensibilité des scintigraphies au disida pour établir un diagnostic d'obstruction était de 50 %. La caractérisation cholangiographique des sténoses a révélé une distribution égale de sténoses lisses (huit sur 13) et irrégulières (cinq sur 13) dans le groupe des sténoses malignes. Dix des 13 sténoses bénignes ont été qualifiées de lisses. Les sténoses malignes étaient nettement plus longues que les sténoses bénignes, soit 30,3 mm par rapport à 9,2 mm (P=0,001). L'analyse des seuils au moyen des courbes ROC a révélé que des sténoses supérieures ou égales à 14 mm étaient un prédicteur de malignité (sensibilité de 78 %, spécificité de 75 %, risque relatif logarithmique de 11,23). CONCLUSIONS : Un niveau de bilirubine sérique de 75 µmol/L ou une longueur de sténose de 14 mm étaient fortement prédictifs de malignité chez les patients atteints d'une sténose biliaire. L'échographie était utile pour prédire les sténoses malignes en décelant soit une dilatation du canal intra-hépatique ou en visualisant la tumeur (primaire ou métastases). Les sténoses d'apparence « bénigne » à la cholangiographie s'avèrent souvent malignes. La scintigraphie au disida n'apportait pas d'informations supplé-mentaires. Une CPRE est nécessaire pour diagnostiquer des sténoses bénignes, qui ont tendance à être moins étendues à l'examen. · Abdominal ultrasound examination with particular attention to intrahepatic biliary dilation, extrahepatic duct calibre, presence or absence of gallbladder and other relevant pathology such as tumour mass or ductal stones. · Disida scan. Patients were examined after a 4 h fast. Opiates were withheld for the proceeding 24 h. In addition to the standard scan, data were collected for deconvolutional analysis to determine hepatic extraction fraction and time activity curve so that the half-life of biliary excretion and time to peak activity could be analyzed. · Cholangiography. ERCP was attempted first in all patients with failures proceeding to PTC. Cefazoline 1 g was administered intravenously 30 to 60 mins before cholangiography. The biliary system was filled as completely as possible using 50% Conray 60 (Mallenchrodt, St Louis, Missouri) contrast injected under low pressure. The information obtained from each cholangiogram included site of stricture, multiplicity, character (smoothly tapered versus irregular or shouldered), stricture(s) length, minimal stricture width, maximal proximal biliary dilation and other information (ampullary mass, primary sclerosing cholangitis, cancer of the pancreas). All data were to be collected within five working days so that the different imaging modalities tested would be comparable. All imaging studies were interpreted by radiologists blinded to the results of the patients' other diagnostic studies. The ERCP data were obtained last so that a biliary stent could be inserted if indicated. The cholangiographic measurements were confirmed by two independent observers. Stricture etiology was defined by cytology or biopsy histology or by clinical outcome after one year. Statistical analysis: Statistical analysis was performed using SPSS. Between-groups differences in mean values of continuous variables were tested by independent samples t tests or by nonparametric Mann-Whitney Rank Sum tests when the distributions were not normal. The differences in frequencies of categorical variables were tested by c 2 test with Yates' correction for continuity or by Fisher's exact test when the expected number of observations per cell was less than five. Associations between continuous variables were assessed by Pearson correlation coefficient. Logistic regression analysis was used to analyze the association of dichotomous outcome variable (malignant versus benign) with continuous and categorical predictor variables. The statistical inferences were based on the level of significance P<0.05. Receiver operating characteristic (ROC) curves were constructed for the biochemical variables. To determine optimal threshold levels for each diagnostic parameter, ROC plots were constructed using the observed true and false positive rates at each potential threshold level. A best fit ROC curve was constructed according to methods published elsewhere (5,6). The threshold value providing the best compromise between true and false positive rates was estimated from the ROC plot. Likelihood ratios were calculated from the fitted ROC curve. RESULTS Thirty-one patients were enrolled in the study. Two patients were excluded -one because he did not have a stricture and one whose suspected stricture was unevaluable because of previous biliary bypass and contraindication for PTC as a result of coagulopathy. Of the remaining 29 patients, 15 were diagnosed with malignant strictures and 14 with benign strictures. Two patients had primary sclerosing cholangitis, both of whom had multiple strictures. Patient demographics and underlying diagnosis are shown i

A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Alcohol, tobacco and breast cancer – collaborative reanalysis of individual data from 53 epidemiological studies, including 58 515 women with breast cancer and 95 067 women without the disease

Alcohol and tobacco consumption are closely correlated and published results on their association with breast cancer have not always allowed adequately for confounding between these exposures. Over 80% of the relevant information worldwide on alcohol and tobacco consumption and breast cancer were collated, checked and analysed centrally. Analyses included 58 515 women with invasive breast cancer and 95 067 controls from 53 studies. Relative risks of breast cancer were estimated, after stratifying by study, age, parity and, where appropriate, women's age when their first child was born and consumption of alcohol and tobacco. The average consumption of alcohol reported by controls from developed countries was 6.0 g per day, i.e. about half a unit/drink of alcohol per day, and was greater in ever-smokers than never-smokers, (8.4 g per day and 5.0 g per day, respectively). Compared with women who reported drinking no alcohol, the relative risk of breast cancer was 1.32 (1.19–1.45, P<0.00001) for an intake of 35–44 g per day alcohol, and 1.46 (1.33–1.61, P<0.00001) for ⩾45 g per day alcohol. The relative risk of breast cancer increased by 7.1% (95% CI 5.5–8.7%; P<0.00001) for each additional 10 g per day intake of alcohol, i.e. for each extra unit or drink of alcohol consumed on a daily basis. This increase was the same in ever-smokers and never-smokers (7.1% per 10 g per day, P<0.00001, in each group). By contrast, the relationship between smoking and breast cancer was substantially confounded by the effect of alcohol. When analyses were restricted to 22 255 women with breast cancer and 40 832 controls who reported drinking no alcohol, smoking was not associated with breast cancer (compared to never-smokers, relative risk for ever-smokers=1.03, 95% CI 0.98–1.07, and for current smokers=0.99, 0.92–1.05). The results for alcohol and for tobacco did not vary substantially across studies, study designs, or according to 15 personal characteristics of the women; nor were the findings materially confounded by any of these factors. If the observed relationship for alcohol is causal, these results suggest that about 4% of the breast cancers in developed countries are attributable to alcohol. In developing countries, where alcohol consumption among controls averaged only 0.4 g per day, alcohol would have a negligible effect on the incidence of breast cancer. In conclusion, smoking has little or no independent effect on the risk of developing breast cancer; the effect of alcohol on breast cancer needs to be interpreted in the context of its beneficial effects, in moderation, on cardiovascular disease and its harmful effects on cirrhosis and cancers of the mouth, larynx, oesophagus and liver

The Cellular Protein MCM3AP Is Required for Inhibition of Cellular DNA Synthesis by the IE86 Protein of Human Cytomegalovirus

Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G(0)-G(1) phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein - an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner