The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer's disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca2+ dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved.
C2 domain; human cognition; KIBRA; membrane binding; phosphatidylinositols
Peri-operative red blood cell transfusions have been associated with post-operative complications in patients undergoing elective orthopaedic hip or knee replacement surgery.
Materials and methods
We performed a post-hoc analysis of data extracted from a randomised study on transfusion triggers using pre-storage leucocyte-depleted red blood cells. Patients who were assigned to the most restrictive transfusion policy ("restrictive group") were compared with patients who were assigned to the most liberal policy ("liberal group"). End-points were red blood cell use, hospital stay, haemoglobin levels, post-operative complications and quality of life scores.
Of 603 patients, 26.4% patients in the restrictive group and 39.1% in the liberal group were transfused (P =0.001). The rate of post-operative infections was lower, although not statistically significantly so, in the restrictive group than in the liberal group (5.4% vs 10.2%, respectively) as was the rate of respiratory complications (1.7% vs 4.9%, respectively), whereas hospital stay, cardiovascular complications and mortality rate were not different in the two groups. Quality of life scores were not associated with type of transfusion policy, the number of red blood cell transfusions or the transfusion status.
A restrictive transfusion protocol was not associated with worse outcome and resulted in a lower transfusion rate compared to the liberal policy. Well-being (quality of life) was not associated with transfusion policy or with red blood cell transfusions.
restrictive transfusion policy; complication rate; orthopaedic surgery; red blood cell transfusion; quality of life
White matter lesions (WML) are clinically relevant since they are associated with strokes, cognitive decline, depression, or epilepsy, but the underlying etiology in young adults without classical risk factors still remains elusive. Our aim was to elucidate the possible clinical diagnosis and mechanisms leading to WML in patients carrying the D313Y mutation in the α-galactosidase A (GLA) gene, a mutation that was formerly described as nonpathogenic. Pathogenic GLA mutations cause Fabry disease, a vascular endothelial glycosphingolipid storage disease typically presenting with a symptom complex of renal, cardiac, and cerebrovascular manifestations. We performed in-depths clinical, biochemical and genetic examinations as well as advanced magnetic resonance imaging analyses in a pedigree with the genetically determined GLA mutation D313Y. We detected exclusive neurologic manifestations of the central nervous system of the “pseudo”-deficient D313Y mutation leading to manifest WML in 7 affected adult family members. Furthermore, two family members that do not carry the mutation showed no WML. The D313Y mutation resulted in a normal GLA enzyme activity in leukocytes and severely decreased activities in plasma. In conclusion, our results provide evidence that GLA D313Y is potentially involved in neural damage with significant WML, demonstrating the necessity of evaluating patients carrying D313Y more thoroughly. D313Y might broaden the spectrum of hereditary small artery diseases of the brain, which preferably occur in young adults without classical risk factors. In view of the existing causal therapy regime, D313Y should be more specifically taken into account in these patients.
The prevalence of hypertension in African Americans (AAs) is higher than in other US groups; yet, few have performed genome-wide association studies (GWASs) in AA. Among people of European descent, GWASs have identified genetic variants at 13 loci that are associated with blood pressure. It is unknown if these variants confer susceptibility in people of African ancestry. Here, we examined genome-wide and candidate gene associations with systolic blood pressure (SBP) and diastolic blood pressure (DBP) using the Candidate Gene Association Resource (CARe) consortium consisting of 8591 AAs. Genotypes included genome-wide single-nucleotide polymorphism (SNP) data utilizing the Affymetrix 6.0 array with imputation to 2.5 million HapMap SNPs and candidate gene SNP data utilizing a 50K cardiovascular gene-centric array (ITMAT-Broad-CARe [IBC] array). For Affymetrix data, the strongest signal for DBP was rs10474346 (P= 3.6 × 10−8) located near GPR98 and ARRDC3. For SBP, the strongest signal was rs2258119 in C21orf91 (P= 4.7 × 10−8). The top IBC association for SBP was rs2012318 (P= 6.4 × 10−6) near SLC25A42 and for DBP was rs2523586 (P= 1.3 × 10−6) near HLA-B. None of the top variants replicated in additional AA (n = 11 882) or European-American (n = 69 899) cohorts. We replicated previously reported European-American blood pressure SNPs in our AA samples (SH2B3, P= 0.009; TBX3-TBX5, P= 0.03; and CSK-ULK3, P= 0.0004). These genetic loci represent the best evidence of genetic influences on SBP and DBP in AAs to date. More broadly, this work supports that notion that blood pressure among AAs is a trait with genetic underpinnings but also with significant complexity.
Background & Aims
Cigarette smoking is an established risk factor for pancreatic cancer, but there is conflicting evidence regarding the effects of alcohol consumption. The effects of cigarettes and alcohol on age of sporadic pancreatic cancer diagnosis have not been examined; we evaluated the independent and synergistic effects of lifetime cigarette smoking and alcohol consumption on age at pancreatic cancer diagnosis in the United States.
We analyzed data on cigarette smoking and alcohol consumption from the IMPAC Medical Registry Services, Cancer Information Resource File (CIRF), collected from June 1, 1993 to December 31, 2003 for 29,239 reported, histologically confirmed cases of pancreatic adenocarcinoma. We also analyzed data on cigarette smoking and alcohol consumption for 820 histologically confirmed cases of pancreatic adenocarcinoma from the University of Michigan Pancreatic Cancer Registry (UMPCR), collected from January 2004 to October 2007.
Current cigarette smokers were diagnosed at significantly younger ages than never smokers, according to data from the CIRF and UMPCR (8.3 years and 6.3 years, respectively); the UMPCR data indicated dose effects. Past and current alcohol consumption were associated with younger age at diagnosis age in both databases. Current smokers who were current drinkers were diagnosed significantly earlier (CIRF 10.2 years, UMPCR 8.6 years) than abstainers. Past cigarette smoking was modestly associated with younger diagnosis age.
Cigarette smoking and alcohol consumption were associated with pancreatic cancer presentation at a younger age among the general population and have a combined effect on diagnosis age. Past cigarette smoking is less influential. Smoking cessation programs could help prevent pancreatic cancer.
Objective To compare postoperative complications in patients undergoing major surgery who received non-filtered or filtered red blood cell transfusions.
Design Prospective, randomised, double blinded trial.
Setting 19 hospitals throughout the Netherlands (three university; 10 clinical; six general).
Participants 1051 evaluable patients: 79 patients with ruptured aneurysm, 412 patients undergoing elective surgery for aneurysm, and 560 undergoing gastrointestinal surgery.
Interventions The non-filtered products had the buffy coat removed and were plasma reduced. The filtered products had the buffy coat removed, were plasma reduced, and filtered before storage to remove leucocytes.
Main outcome measures Mortality and duration of stay in intensive care. Secondary end points were occurrence of multi-organ failure, infections, and length of hospital stay.
Results No significant differences were found in mortality (odds ratio for filtered v non-filtered 0.80, 95% confidence interval 0.53 to 1.21) and in mean stay in intensive care (- 0.4 day, - 1.6 to 0.6 day). In the filtered group the mean length of hospital stay was 2.4 days shorter (- 4.8 to 0.0 day; P = 0.050) and the incidence of multi-organ failure was 30% lower (odds ratio 0.70, 0.49 to 1.00; P = 0.050). There were no differences in rates of infection (0.98, 0.73 to 1.32).
Conclusion The use of filtered transfusions in some types of major surgery may reduce the length of hospital stay and the incidence of postoperative multi-organ failure.
The post-hospital care of 290 patients with selected chronic conditions of a specific severity who were discharged over a 3-month period from a general hospital in Halifax, Canada, was studied. The majority of the patients were married. The average age of the men was 59.2 years and of the women 58.1. More than half of the patients belonged to the low socioeconomic group earning between $1,000 and $6,999 a year. Their average period of education was 8.4 years. Interviews with the patients about their compliance with physicians' orders revealed that 40.4 percent had not complied with one or more of their physician's recommendations. Lack of compliance was related to age, marital status, education, income, and severity of disease. It was also associated with high dosages of medicine and multiple prescriptions. Cost barriers constituted a significant factor in noncompliance.
Mammographic density is a strong risk factor for breast cancer. Apart from hormone replacement therapy (HRT), little is known about lifestyle factors that influence breast density.
We examined the effect of smoking, alcohol and physical activity on mammographic density in a population-based sample of postmenopausal women without breast cancer. Lifestyle factors were assessed by a questionnaire and percentage and area measures of mammographic density were measured using computer-assisted software. General linear models were used to assess the association between lifestyle factors and mammographic density and effect modification by body mass index (BMI) and HRT was studied.
Overall, alcohol intake was positively associated with percent mammographic density (P trend = 0.07). This association was modified by HRT use (P interaction = 0.06): increasing alcohol intake was associated with increasing percent density in current HRT users (P trend = 0.01) but not in non-current users (P trend = 0.82). A similar interaction between alcohol and HRT was found for the absolute dense area, with a positive association being present in current HRT users only (P interaction = 0.04). No differences in mammographic density were observed across categories of smoking and physical activity, neither overall nor in stratified analyses by BMI and HRT use.
Increasing alcohol intake is associated with an increase in mammography density, whereas smoking and physical activity do not seem to influence density. The observed interaction between alcohol and HRT may pose an opportunity for HRT users to lower their mammographic density and breast cancer risk.
genetic association study; disease genetics; immunogenetics; liver
Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P2 modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P2 dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.
Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.
Heparan sulfate Proteoglycans (HSPG) are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG’s, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.
The European Union (EU) health mandate was initially defined in the Maastricht Treaty in 1992. The twentieth anniversary of the Treaty offers a unique opportunity to take stock of EU health actions by giving an overview of influential public health related EU-level policy outputs and a summary of policy outputs or actions perceived as an achievement, a failure or a missed opportunity.
Semi-structured expert interviews (N = 20) were conducted focusing on EU-level actions that were relevant for health. Respondents were asked to name EU policies or actions that they perceived as an achievement, a failure or a missed opportunity. A directed content analysis approach was used to identify expert perceptions on achievements, failures and missed opportunities in the interviews. Additionally, a nominal group technique was applied to identify influential and public health relevant EU-level policy outputs.
The ranking of influential policy outputs resulted in top positions of adjudications and legislations, agencies, European Commission (EC) programmes and strategies, official networks, cooperative structures and exchange efforts, the work on health determinants and uptake of scientific knowledge. The assessment of EU health policies as being an achievement, a failure or a missed opportunity was often characterized by diverging respondent views. Recurring topics that emerged were the Directorate General for Health and Consumers (DG SANCO), EU agencies, life style factors, internal market provisions as well as the EU Directive on patients’ rights in cross-border healthcare. Among these recurring topics, expert perceptions on the establishment of DG SANCO, EU public health agencies, and successes in tobacco control were dominated by aspects of achievements. The implementation status of the Health in All Policy approach was perceived as a missed opportunity.
When comparing the emerging themes from the interviews conducted with the responsibilities defined in the EU health mandate, one can identify that these responsibilities were only partly fulfilled or acknowledged by the respondents. In general, the EU is a recognized public health player in Europe which over the past two decades, has begun to develop competencies in supporting, coordinating and supplementing member state health actions. However, the assurance of health protection in other European policies seems to require further development.
European Union; EU health mandate; Health policy; Assessment; Qualitative research
Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS) as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs) from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total) tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC) including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3.
DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs) regarding inflammatory bowel disease (IBD) susceptibility and examined their functional impact on transcription factor binding and downstream gene expression.
Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn’s disease (CD) patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays.
IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0×10−7, OR 1.42; 95% CI 1.24–1.63). All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1×10−18). The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.
We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important role of DMBT1 in CD pathogenesis.
Individual sites of superoxide production in the mitochondrial respiratory chain have previously been defined and partially characterized using specific inhibitors, but the native contribution of each site to total superoxide production in the absence of inhibitors is unknown. We estimated rates of superoxide production (measured as H2O2) at different sites in rat muscle mitochondria using specific endogenous reporters. The rate of superoxide production by the complex I flavin (site IF) was calibrated to the reduction state of endogenous NAD(P)H. Similarly, the rate of superoxide production by the complex III site of quinol oxidation (site IIIQo) was calibrated to the reduction state of endogenous cytochrome b566. We then measured the endogenous reporters in mitochondria oxidizing NADH-generating substrates, without added respiratory inhibitors, with and without ATP synthesis. We used the calibrated reporters to calculate the rates of superoxide production from sites IF and IIIQo. The calculated rates of superoxide production accounted for much of the measured overall rates. During ATP synthesis, site IF was the dominant superoxide producer. Under non-phosphorylating conditions, overall rates were higher and sites IF, IIIQo and unidentified sites (perhaps the complex I site of quinone reduction, site IQ) all made substantial contributions to measured H2O2 production.
mitochondria; ROS; superoxide; complex I; complex III; NADH autofluorescence; cytochrome b.
Pancreatic cancer remains one of the most challenging of all cancers. Genetic risk factors are believed to play a major role, but other than genes coding for blood group, genetic risks for sporadic cases remain elusive. However, several germline mutations have been identified that lead to hereditary pancreatic cancer, familial pancreatic cancer and increased risk for pancreatic cancer as part of a familial cancer syndrome. The most important genes with variants increasing risk for pancreatic cancer include BRCA1, BRCA2, PALB2, ATM, CDKN2A, APC, MLH1, MSH2, MSH6, PMS2, PRSS1 and STK11. Recognition of members of high-risk families is important for understanding pancreatic cancer biology, for recommending risk reduction strategies and, in some cases, initiating cancer surveillance programs. Because the best methods for surveillance have not been established the recommendation to refer at-risk patients to centers with ongoing research programs in pancreatic cancer surveillance is supported.
Pancreatic cancer; genetic risk factors; germline mutations
High-throughput mass spectrometry methods have been developed to screen newborns for lysosomal storage disorders, allowing the implementation of newborn screening pilot studies in North America and Europe. It is currently feasible to diagnose Pompe, Fabry, Gaucher, Krabbe, and Niemann-Pick A/B diseases, as well as mucopolysaccharidosis I, by tandem mass spectrometry in dried blood spots, which offers considerable technical advantages compared with standard methodologies. We aimed to investigate whether the mass spectrometry methodology for lysosomal storage disease screening, originally developed for newborns, can also discriminate between affected patients and controls of various ages.
A total of 205 control individuals were grouped according to age and subjected to mass spectrometry quantification of lysosomal α-glucosidase, β-glucocerebrosidase, α-galactosidase, acid sphingomyelinase, galactocerebrosidase, and α−L-iduronidase activities. Additionally, 13 affected patients were analyzed.
The median activities for each enzyme and each age group were determined. Enzyme activities were significantly lower in individuals aged older than 18 years compared with those in newborns. Affected patients presented enzymatic activities corresponding to less than 20% of the age-matched controls.
Our data indicate that the mass spectrometry methodology can be used for the screening of lysosomal storage diseases in non-newborn patients. However, for some diseases, such as Fabry and mucopolysaccharidosis I, a combination of biochemical and clinical data may be necessary to achieve accurate diagnoses.
Dried Blood Spot Analysis; Pompe Disease; Fabry Disease; Gaucher Disease; Krabbe Disease