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1.  Design and baseline characteristics of the Food4Me study: a web-based randomised controlled trial of personalised nutrition in seven European countries 
Genes & Nutrition  2014;10(1):450.
Improving lifestyle behaviours has considerable potential for reducing the global burden of non-communicable diseases, promoting better health across the life-course and increasing well-being. However, realising this potential will require the development, testing and implementation of much more effective behaviour change interventions than are used conventionally. Therefore, the aim of this study was to conduct a multi-centre, web-based, proof-of-principle study of personalised nutrition (PN) to determine whether providing more personalised dietary advice leads to greater improvements in eating patterns and health outcomes compared to conventional population-based advice. A total of 5,562 volunteers were screened across seven European countries; the first 1,607 participants who fulfilled the inclusion criteria were recruited into the trial. Participants were randomly assigned to one of the following intervention groups for a 6-month period: Level 0—control group—receiving conventional, non-PN advice; Level 1—receiving PN advice based on dietary intake data alone; Level 2—receiving PN advice based on dietary intake and phenotypic data; and Level 3—receiving PN advice based on dietary intake, phenotypic and genotypic data. A total of 1,607 participants had a mean age of 39.8 years (ranging from 18 to 79 years). Of these participants, 60.9 % were women and 96.7 % were from white-European background. The mean BMI for all randomised participants was 25.5 kg m−2, and 44.8 % of the participants had a BMI ≥ 25.0 kg m−2. Food4Me is the first large multi-centre RCT of web-based PN. The main outcomes from the Food4Me study will be submitted for publication during 2015.
Electronic supplementary material
The online version of this article (doi:10.1007/s12263-014-0450-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s12263-014-0450-2
PMCID: PMC4261071  PMID: 25491748
Personalised nutrition; Web-based; Phenotype; Genotype; Randomised controlled trial
2.  Susceptibility-Weighted Angiography of Intracranial Blood Products and Calcifications Compared to Gradient Echo Sequence 
The neuroradiology journal  null;26(5):493-500.
Summary
This study evaluated the sensitivity of susceptibility-weighted angiography (SWAN) compared with gradient echo (GRE) sequence in the depiction of haemorrhagic and calcium lesions by virtue of correlation analysis between the number, area and contrast index. The study included 21 patients (15 women, 6 men; age range 18 to 80 years) in whom intracranial haemorrhage or calcifications were previously diagnosed with CT and/or MR. GRE and SWAN sequences were performed as part of a conventional Brain MR study using a 3T scanner. Pathologic findings were: cavernoma (n=8), chronic intraparenchymal haemorrhage (n=5), petechial bleeding (n=3), parenchymal calcifications (n=2), sequelae of haemorrhagic contusion focus (n=1), post-surgical glioma (n=1) and venous angioma (n=1). In eight patients, more lesions were found in the SWAN sequence than in GRE. In the measurement of the largest area, in all cases the measured area was larger in the SWAN sequence than in GRE. The SWAN sequence was reported to have shown higher contrast between the lesion and the healthy white matter than in GRE. The SWAN sequence is more sensitive than GRE in the identification of cerebral haemorrhage and calcifications. The SWAN sequence also obtained significantly larger images than with GRE, and a higher contrast difference between the lesion and the healthy parenchyma.
PMCID: PMC4202827  PMID: 24199808
MRI scans; brain imaging; intracerebral haemorrhage; cerebral calcification; cerebral cavernous malformations
3.  Effects of Exercise Modalities on Arterial Stiffness and Wave Reflection: A Systematic Review and Meta-Analysis of Randomized Controlled Trials 
PLoS ONE  2014;9(10):e110034.
Background and Objectives
Physical activity is associated with lower cardiovascular and all-cause mortality. However, the effects of different exercise modalities on arterial stiffness are currently unclear. Our objectives were to investigate the effects of exercise modalities (aerobic, resistance or combined) on pulse wave velocity (PWV) and augmentation index (AIx), and to determine whether the effects on these indices differed according to the participants' or exercise characteristics.
Methods
We searched the Medline, Embase and Cochrane Library databases from inception until April 2014 for randomized controlled trials lasting ≥4 weeks investigating the effects of exercise modalities on PWV and AIx in adults aged ≥18 years.
Results
Forty-two studies (1627 participants) were included in this analysis. Aerobic exercise improved both PWV (WMD: −0.63 m/s, 95% CI: −0.90, −0.35) and AIx (WMD:−2.63%, 95% CI: −5.25 to −0.02) significantly. Aerobic exercise training showed significantly greater reduction in brachial-ankle (WMD: −1.01 m/s, 95% CI: −1.57, −0.44) than in carotid-femoral (WMD: -0.39 m/s, 95% CI: −0.52, −0.27) PWV. Higher aerobic exercise intensity was associated with larger reductions in AIx (β: −1.55%, CI −3.09, 0.0001). In addition, aerobic exercise had a significantly larger effect in reducing PWV (WMD:−1.0 m/s, 95% CI: −1.43, −0.57) in participants with stiffer arteries (PWV ≥8 m/s). Resistance exercise had no effect on PWV and AIx. There was no significant effect of combined exercise on PWV and AIx.
Conclusions
We conclude that aerobic exercise improved arterial stiffness significantly and that the effect was enhanced with higher aerobic exercise intensity and in participants with greater arterial stiffness at baseline.
Trial Registration PROSPERO
Database registration: CRD42014009744,.
doi:10.1371/journal.pone.0110034
PMCID: PMC4198209  PMID: 25333969
4.  Thromboprophylaxis for trauma patients 
This is the protocol for a review and there is no abstract. The objectives are as follows:
To assess the effects of thromboprophylaxis in trauma patients on mortality and incidence of DVT and PE.
To compare the effects of different thromboprophylaxis interventions and their relative effects according to the type of trauma.
doi:10.1002/14651858.CD008303
PMCID: PMC4176629  PMID: 25267908
6.  Should Physical Activity Recommendations Be Ethnicity-Specific? Evidence from a Cross-Sectional Study of South Asian and European Men 
PLoS ONE  2013;8(12):e82568.
Background
Expert bodies and health organisations recommend that adults undertake at least 150 min.week−1 of moderate-intensity physical activity (MPA). However, the underpinning data largely emanate from studies of populations of European descent. It is unclear whether this level of activity is appropriate for other ethnic groups, particularly South Asians, who have increased cardio-metabolic disease risk compared to Europeans. The aim of this study was to explore the level of MPA required in South Asians to confer a similar cardio-metabolic risk profile to that observed in Europeans undertaking the currently recommended MPA level of 150 min.week−1.
Methods
Seventy-five South Asian and 83 European men, aged 40–70, without cardiovascular disease or diabetes had fasted blood taken, blood pressure measured, physical activity assessed objectively (using accelerometry), and anthropometric measures made. Factor analysis was used to summarise measured risk biomarkers into underlying latent ‘factors’ for glycaemia, insulin resistance, lipid metabolism, blood pressure, and overall cardio-metabolic risk. Age-adjusted regression models were used to determine the equivalent level of MPA (in bouts of ≥10 minutes) in South Asians needed to elicit the same value in each factor as Europeans undertaking 150 min.week−1 MPA.
Findings
For all factors, except blood pressure, equivalent MPA values in South Asians were significantly higher than 150 min.week−1; the equivalent MPA value for the overall cardio-metabolic risk factor was 266 (95% CI 185-347) min.week−1.
Conclusions
South Asian men may need to undertake greater levels of MPA than Europeans to exhibit a similar cardio-metabolic risk profile, suggesting that a conceptual case can be made for ethnicity-specific physical activity guidance. Further study is needed to extend these findings to women and to replicate them prospectively in a larger cohort.
doi:10.1371/journal.pone.0082568
PMCID: PMC3859604  PMID: 24349313
7.  The Intracellular Domain of Sortilin Interacts with Amyloid Precursor Protein and Regulates Its Lysosomal and Lipid Raft Trafficking 
PLoS ONE  2013;8(5):e63049.
The processing of Amyloid precursor protein (APP) is multifaceted, comprising of protein transport, internalization and sequential proteolysis. However, the exact mechanism of APP intracellular trafficking and distribution remains unclear. To determine the interaction between sortilin and APP and the effect of sortilin on APP trafficking and processing, we studied the binding site and its function by mapping experiments, colocalization, coimmunoprecipitation and sucrose gradient fractionation. We identified for the first time that sortilin interacts with APP at both N- and C-terminal regions. The sortilin-FLVHRY (residues 787–792) and APP-NPTYKFFE (residues 759–766) motifs are crucial for the C-terminal interaction. We also found that lack of the FLVHRY motif reduces APP lysosomal targeting and increases APP distribution in lipid rafts in co-transfected HEK293 cells. These results are consistent with our in vivo data where sortilin knockout mice showed a decrease of APP lysosomal distribution and an increase of APP in lipid rafts. We further confirmed that overexpression of sortilin-FLVHRY mutants failed to rescue the lysosomal degradation of APP. Thus, our data suggests that sortilin is implicated in APP lysosomal and lipid raft targeting via its carboxyl-terminal F/YXXXXF/Y motif. Our study provides new molecular insights into APP trafficking and processing.
doi:10.1371/journal.pone.0063049
PMCID: PMC3660575  PMID: 23704887
8.  Expression of Eag1 K+ channel and ErbBs in human pituitary adenomas: cytoskeleton arrangement patterns in cultured cells 
Pituitary adenomas can invade surrounded tissue, but the mechanism remains elusive. Ether à go-go-1 (Eag1) potassium channel and epidermal growth factor receptors (ErbB1 and ErbB2) have been associated to invasive phenotypes or poor prognosis in cancer patients. However, cells arrange their cytoskeleton in order to acquire a successful migration pattern. We have studied ErbBs and Eag1 expression, and cytoskeleton arrangements in 11 human pituitary adenomas. Eag1, ErbB1 and ErbB2 expression were studied by immunochemistry in tissue and cultured cells. The cytoskeleton arrangement was analyzed in cultured cells by immunofluorescence. Normal pituitary tissue showed ErbB2 expression and Eag1 only in few cells. However, Eag1 and ErbB2 were expressed in all the tumors analyzed. ErbB1 expression was observed variable and did not show specificity for a tumor characteristic. Cultured cells from micro- and macro-adenomas clinically functional organize their cytoskeleton suggesting a mesenchymal pattern, and a round leucocyte/amoeboid pattern from invasive clinically silent adenoma. Pituitary tumors over-express EGF receptors and the ErbB2 repeated expression suggests is a characteristic of adenomas. Eag 1 was express, in different extent, and could be a therapeutic target. The cytoskeleton arrangements observed suggest that pituitary tumor cells acquire different patterns: mesenchymal, and leucocyte/amoeboid, the last observed in the invasive adenomas. Amoeboid migration pattern has been associated with high invasion capacity.
PMCID: PMC3563198  PMID: 23413122
Pituitary adenomas; ErbBs; Eag1; cytoskeleton; invasion; cell culture
9.  Hepatic sortilin regulates both apolipoprotein B secretion and LDL catabolism 
The Journal of Clinical Investigation  2012;122(8):2807-2816.
Genome-wide association studies (GWAS) have identified a genetic variant at a locus on chromosome 1p13 that is associated with reduced risk of myocardial infarction, reduced plasma levels of LDL cholesterol (LDL-C), and markedly increased expression of the gene sortilin-1 (SORT1) in liver. Sortilin is a lysosomal sorting protein that binds ligands both in the Golgi apparatus and at the plasma membrane and traffics them to the lysosome. We previously reported that increased hepatic sortilin expression in mice reduced plasma LDL-C levels. Here we show that increased hepatic sortilin not only reduced hepatic apolipoprotein B (APOB) secretion, but also increased LDL catabolism, and that both effects were dependent on intact lysosomal targeting. Loss-of-function studies demonstrated that sortilin serves as a bona fide receptor for LDL in vivo in mice. Our data are consistent with a model in which increased hepatic sortilin binds intracellular APOB-containing particles in the Golgi apparatus as well as extracellular LDL at the plasma membrane and traffics them to the lysosome for degradation. We thus provide functional evidence that genetically increased hepatic sortilin expression both reduces hepatic APOB secretion and increases LDL catabolism, providing dual mechanisms for the very strong association between increased hepatic sortilin expression and reduced plasma LDL-C levels in humans.
doi:10.1172/JCI63563
PMCID: PMC3408750  PMID: 22751103
10.  Impact of patient-prosthesis mismatch on 30-day outcomes in young and middle-aged patients undergoing aortic valve replacement 
Background
The impact of patient-prosthesis mismatch (PPM) on early outcomes in young and middle-aged patients undergoing conventional aortic valve replacement for severe aortic stenosis remains unknown. Our objective was to evaluate the incidence of some degree of PPM and its influence on early mortality and morbidity.
Methods
We analyzed our single center experience in all patients <70 years undergoing first-time isolated aortic valve replacement for severe stenosis in our center from September 2007 to September 2011. PPM was defined as an indexed effective orifice area ≤ 0,85 cm2/m2. The influence of PPM on early mortality and postoperative complications was studied using propensity score analysis. Follow up at 30 postoperative days was 100% complete.
Results
Of 199 patients studied, 61 (30,7%) had some degree of PPM. PPM was associated with an increased postoperative mortality (OR = 8,71; 95% CI = 1,67–45,29; p = 0,04) and major postoperative complications (OR = 2,96; CI = 1,03–8,55; p = 0,044). However, no association between PPM and prolonged hospital or ICU stay was demonstrated.
Conclusions
Moderate PPM is a common finding in young and middle-aged patients undergoing surgery for aortic valve replacement due to severe stenosis. In addition, its influence on early outcomes may be relevant.
doi:10.1186/1749-8090-7-46
PMCID: PMC3428658  PMID: 22587627
Patient-prosthesis mismatch; Severe aortic stenosis
11.  Objective vs. Self-Reported Physical Activity and Sedentary Time: Effects of Measurement Method on Relationships with Risk Biomarkers 
PLoS ONE  2012;7(5):e36345.
Purpose
Imprecise measurement of physical activity variables might attenuate estimates of the beneficial effects of activity on health-related outcomes. We aimed to compare the cardiometabolic risk factor dose-response relationships for physical activity and sedentary behaviour between accelerometer- and questionnaire-based activity measures.
Methods
Physical activity and sedentary behaviour were assessed in 317 adults by 7-day accelerometry and International Physical Activity Questionnaire (IPAQ). Fasting blood was taken to determine insulin, glucose, triglyceride and total, LDL and HDL cholesterol concentrations and homeostasis model-estimated insulin resistance (HOMAIR). Waist circumference, BMI, body fat percentage and blood pressure were also measured.
Results
For both accelerometer-derived sedentary time (<100 counts.min−1) and IPAQ-reported sitting time significant positive (negative for HDL cholesterol) relationships were observed with all measured risk factors – i.e. increased sedentary behaviour was associated with increased risk (all p≤0.01). However, for HOMAIR and insulin the regression coefficients were >50% lower for the IPAQ-reported compared to the accelerometer-derived measure (p<0.0001 for both interactions). The relationships for moderate-to-vigorous physical activity (MVPA) and risk factors were less strong than those observed for sedentary behaviours, but significant negative relationships were observed for both accelerometer and IPAQ MVPA measures with glucose, and insulin and HOMAIR values (all p<0.05). For accelerometer-derived MVPA only, additional negative relationships were seen with triglyceride, total cholesterol and LDL cholesterol concentrations, BMI, waist circumference and percentage body fat, and a positive relationship was evident with HDL cholesterol (p = 0.0002). Regression coefficients for HOMAIR, insulin and triglyceride were 43–50% lower for the IPAQ-reported compared to the accelerometer-derived MVPA measure (all p≤0.01).
Conclusion
Using the IPAQ to determine sitting time and MVPA reveals some, but not all, relationships between these activity measures and metabolic and vascular disease risk factors. Using this self-report method to quantify activity can therefore underestimate the strength of some relationships with risk factors.
doi:10.1371/journal.pone.0036345
PMCID: PMC3348936  PMID: 22590532
12.  Synthesis and characterization of biocompatible-nanohydroxyapatite crystals obtained by a modified sol-gel processing 
Biomatter  2012;2(2):71-76.
A modified sol-gel process for synthesizing nanocrystalline hydroxyapatite powders (nHA) for biomedical applications, using tetrahydrated calcium nitrate [Ca(NO3)2∙4H2O] and phosphorous pentoxide [P2O5] as precursor, is presented and discussed. The powders were washed and heat-treated at different temperatures and then characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The total process time reached with this modified process was less than 16 h. The results showed that there was an increment in size of the HA nanocrystals (nHA) when treated at different temperatures, ranging from 30 nm for the sample treated at 600°C to 500 nm for the sample heat-treated at 1200°C.
doi:10.4161/biom.20379
PMCID: PMC3549859  PMID: 23507804
X-ray diffraction; biomaterials; hydroxyapatite; nanocrystals; sol-gel
13.  Insulin Resistance in Chileans of European and Indigenous Descent: Evidence for an Ethnicity x Environment Interaction 
PLoS ONE  2011;6(9):e24690.
Background
Effects of urbanisation on diabetes risk appear to be greater in indigenous populations worldwide than in populations of European origin, but the reasons are unclear. This cross-sectional study aimed to determine whether the effects of environment (Rural vs. Urban), adiposity, fitness and lifestyle variables on insulin resistance differed between individuals of indigenous Mapuche origin compared to those of European origin in Chile.
Methodology/Principal Findings
123 Rural Mapuche, 124 Urban Mapuche, 91 Rural European and 134 Urban European Chilean adults had blood taken for determination of HOMA-estimated insulin resistance (HOMAIR) and underwent assessment of physical activity/sedentary behaviour (using accelerometry), cardiorespiratory fitness, dietary intake and body composition. General linear models were used to determine interactions with ethnicity for key variables. There was a significant “ethnicity x environment” interaction for HOMAIR (Mean±SD; Rural Mapuche: 1.65±2.03, Urban Mapuche: 4.90±3.05, Rural European: 0.82±0.61, Urban European: 1.55±1.34, p(interaction) = 0.0003), such that the effect of urbanisation on HOMAIR was greater in Mapuches than Europeans. In addition, there were significant interactions (all p<0.004) with ethnicity for effects of adiposity, sedentary time and physical activity on HOMAIR, with greater effects seen in Mapuches compared to Europeans, an observation that persisted after adjustment for potential confounders.
Conclusions/Significance
Urbanisation, adiposity, physical activity and sedentary behaviour influence insulin resistance to a greater extent in Chilean Mapuches than Chileans of European descent. These findings have implications for the design and implementation of lifestyle strategies to reduce metabolic risk in different ethnic groups, and for understanding of the mechanisms underpinning human insulin resistance.
doi:10.1371/journal.pone.0024690
PMCID: PMC3169638  PMID: 21931814
14.  From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus 
Nature  2010;466(7307):714-719.
Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 as strongly associated with both serum low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP transcription factor binding site and alters the hepatic expression of the SORT1 gene. With siRNA knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.
doi:10.1038/nature09266
PMCID: PMC3062476  PMID: 20686566
15.  Surgical site infection in abdominal trauma patients: risk prediction and performance of the NNIS and SENIC indexes 
Canadian Journal of Surgery  2011;54(1):17-24.
Background
The National Nosocomial Infections Surveillance (NNIS) and Efficacy of Nosocomial Infection Control (SENIC) indexes are designed to develop control strategies and to reduce morbidity and mortality rates resulting from infections in surgical patients. We sought to assess the application of these indexes in patients under-going surgery for abdominal trauma and to develop an alternative model to predict surgical site infections (SSIs).
Methods
We conducted a prospective cohort study between November 2000 and March 2002. The main outcome measure was SSIs. We evaluated the variables included in the NNIS and SENIC indexes and some preoperative, intraoperative and postoperative variables that could be risk factors related to the development of SSIs. We performed multivariate analyses using a forward logistic regression method. Finally, we assessed infection risk prediction, comparing the estimated probabilities with actual occurrence using the areas under the receiver operating characteristic (ROC) curves.
Results
Overall, 614 patients underwent an exploratory laparotomy. Of these, 85 (13.8%) experienced deep incisional and organ/intra-abdominal SSIs. The independent variables associated with this complication were an Abdominal Trauma Index score greater than 24, abdominal contamination and admission to the intensive care unit. We proposed a model for predicting deep incisional and organ/intra-abdominal SSIs using these variables (alternative model). The areas under the ROC curves were compared using the estimated probabilities for this alternative model and for the NNIS and SENIC scores. The analysis revealed a greater area under the ROC curve for the alternative model. The NNIS and SENIC scores did not perform as well as the alternative model in patients with abdominal trauma.
Conclusion
The NNIS and SENIC indexes were inferior to the proposed alternative model for predicting SSIs in patients undergoing surgery for abdominal trauma.
doi:10.1503/cjs.022109
PMCID: PMC3038362  PMID: 21251428
16.  ATP-Binding Cassette Transporters ABCA1, ABCA7 and ABCG1 in Mouse Spermatozoa 
Mammalian spermatozoa lose plasma membrane cholesterol during their maturation in the epididymis and during their capacitation in the female reproductive tract. While acceptors such as high-density lipoproteins (HDL) and apolipoproteins A-I (apoA-I) and J have been found in male and female reproductive tracts, transporters that mediate cholesterol efflux from plasma membranes of spermatozoa to such acceptors have not yet been defined. Candidate transporters are members of the ATP-binding cassette (ABC) transporter superfamily including ABCA1, ABCA7, ABCG1 and ABCG4, which have all been implicated in the transport of sterols and phospholipids to apolipoproteins and HDL. Here we show that mouse spermatozoa in the seminiferous tubules and epididymis express ABCA1, ABCA7 and ABCG1, but not ABCG4. Moreover, we show that ABCA1, ABCA7 and ABCG1 antibodies decrease cholesterol efflux from spermatozoa to lipid acceptors apoA-I and albumin and inhibit in vitro fertilization.
doi:10.1016/j.bbrc.2008.09.009
PMCID: PMC2976870  PMID: 18793613
ATP-binding cassette transporters; cholesterol efflux; in vitro fertilization; sperm; mouse
17.  Mice Doubly-Deficient in Lysosomal Hexosaminidase A and Neuraminidase 4 Show Epileptic Crises and Rapid Neuronal Loss 
PLoS Genetics  2010;6(9):e1001118.
Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts GM2 to GM3 ganglioside. Hexa−/− mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise GM2 ganglioside via a lysosomal sialidase into glycolipid GA2, which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4−/−;Hexa−/−) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa−/− or Neu4−/− siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating GM2 ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa−/− mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa−/− mice.
Author Summary
Tay-Sachs disease is the second most common lysosomal storage disorder, especially frequent in Ashkenazi Jews and French Canadians. The disorder is caused by mutations in the gene coding for lysosomal β-hexosaminidase A (HexA), resulting in accumulation of GM2 ganglioside in neurons followed by progressive neurologic degeneration, fatal in early childhood. However mice, depleted of HexA, remain asymptomatic to at least 1 year of age, owing to the ability of these mice to catabolise stored GM2 ganglioside via a lysosomal neuraminidase into glycolipid GA2 further processed by β-hexosaminidase B, thereby completely bypassing the HexA defect. Our current study provides an explanation why the disease is severe in humans but not in mice. We showed that mice depleted of both HexA and ganglioside neuraminidase 4 (Neu4) show epileptic seizures similar to that often observed in Tay-Sachs patients. Single HexA or Neu4 knockout mice do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating cortical and hippocampal neurons and multiple layers of cortical neurons accumulating GM2 ganglioside. Our data suggest that the Neu4 depletion exacerbates the disease in HexA knockout mice, supporting the view that Neu4 is one of the modifier genes in the mouse model of Tay-Sachs disease.
doi:10.1371/journal.pgen.1001118
PMCID: PMC2940724  PMID: 20862357
18.  Editing of CD1d-Bound Lipid Antigens by Endosomal Lipid Transfer Proteins 
Science (New York, N.Y.)  2003;303(5657):523-527.
It is now established that CD1 molecules present lipid antigens to T cells, although it is not clear how the exchange of lipids between membrane compartments and the CD1 binding groove is assisted. We report that mice deficient in prosaposin, the precursor to a family of endosomal lipid transfer proteins (LTP), exhibit specific defects in CD1d-mediated antigen presentation and lack Vα14 NKT cells. In vitro, saposins extracted monomeric lipids from membranes and from CD1, thereby promoting the loading as well as the editing of lipids on CD1. Transient complexes between CD1, lipid, and LTP suggested a “tug-of-war” model in which lipid exchange between CD1 and LTP is on the basis of their respective affinities for lipids. LTPs constitute a previously unknown link between lipid metabolism and immunity and are likely to exert a profound influence on the repertoire of self, tumor, and microbial lipid antigens.
doi:10.1126/science.1092009
PMCID: PMC2918537  PMID: 14684827 CAMSID: cams1387
19.  A randomized clinical trial of unfractioned heparin for treatment of sepsis (the HETRASE study): design and rationale [NCT00100308] 
Trials  2006;7:19.
Introduction
Infection promotes coagulation via a large number of molecular and cellular mechanisms, and this procoagulant activity has boosted basic and clinical research using anticoagulant molecules as therapeutic tools in sepsis. Heparin, which is a naturally occurring proteoglycan that acts by reducing thrombin generation and fibrin formation, has not been rigorously tested in a randomized clinical trial.
Methods
Randomized, double-masked, placebo-controlled, single-center clinical trial. Patients are recruited through the emergency room at Hospital Universitario San Vicente de Paul. This is a 650-bed University Hospital in Medellín, Colombia and is a referral center for a region with approximately 3 million habitants. The recruitment process started on July 2005 and will finish on June 2007. Patients aged 18 years or older, males or females, hospitalized with clinically or microbiological confirmed sepsis, have been included. The interventions are unfractioned heparin in low dose continuous infusion (500 units per hour for 7 days) or placebo, additionally to the standard of care for sepsis patients in Colombia.
Results
Our primary aims are to estimate the effects of heparin on hospital length of stay and change from baseline Multiple Organ Dysfunction (MOD) score. Secondary objectives are to estimate the effects of heparin on 28-day all-cause mortality, and to estimate the possible effect modification on 28-day all-cause mortality, in subgroups defined by source and site of infection, and baseline values of APACHE II score, MOD score and D-dimer.
Conclusion
The available literature in animal and human research, and the understanding of the molecular biology regarding inflammation and coagulation, supports a randomized clinical trial for the use of heparin in sepsis. Our study will provide appropriate power to detect differences in valid surrogate outcomes, and it will explore important preliminary data for efficacy regarding the clinical end-point of mortality.
doi:10.1186/1745-6215-7-19
PMCID: PMC1482716  PMID: 16729879
21.  Spam1-associated transmission ratio distortion in mice: Elucidating the mechanism 
Background
While transmission ratio distortion, TRD, (a deviation from Mendelian ratio) is extensive in humans and well-documented in mice, the underlying mechanisms are unknown. Our earlier studies on carriers of spontaneous mutations of mouse Sperm Adhesion Molecule 1 (Spam1) suggested that TRD results from biochemically different sperm, due to a lack of transcript sharing through the intercellular cytoplasmic bridges of spermatids. These bridges usually allow transcript sharing among genetically different spermatids which develop into biochemically and functionally equivalent sperm.
Objectives
The goals of the study were to provide support for the lack of sharing (LOS) hypothesis, using transgene and null carriers of Spam1, and to determine the mechanism of Spam1-associated TRD.
Methods
Carriers of Spam1-Hyal5 BAC transgenes were mated with wild-type female mice and the progeny analyzed for TRD by PCR genotyping. Sperm from transgene and Spam1 null carriers were analyzed using flow cytometry and immunocytochemistry to detect quantities of Spam1 and/or Hyal5. Transgene-bearing sperm with Spam1 overexpression were detected by fluorescence in situ hybridization. In wild-type animals, EM studies of in situ transcript hybridization of testis sections and Northern analysis of biochemically fractionated testicular RNA were performed to localize Spam1 transcript. Finally, AU-rich motifs identified in the 3' UTR of Spam1 RNA were assayed by UV cross-linking to determine their ability to interact with testicular RNA binding proteins.
Results
The Tg8 line of transgene carriers had a significant (P < 0.001) TRD, due to reduced fertilizing ability of transgene-bearing sperm. These sperm retained large cytoplasmic droplets engorged with overexpressed Spam1 or Hyal5 protein. Caudal sperm from transgene carriers and caput sperm of null carriers showed a bimodal distribution of Spam1, indicating that the sperm in a male were biochemically different with respect to Spam1 quantities. Spam1 RNA was absent from the bridges, associated exclusively with the ER, and was shown to be anchored to the cytoskeleton. This compartmentalization of the transcript, mediated by cytoskeletal binding, occurs via protein interactions with 3' UTR AU-rich sequences that are likely involved in its stabilization.
Conclusion
We provide strong support for the LOS hypothesis, and have elucidated the mechanism of Spam1-associated TRD.
doi:10.1186/1477-7827-3-32
PMCID: PMC1201170  PMID: 16092963
22.  Mice Lacking the UBC4-testis Gene Have a Delay in Postnatal Testis Development but Normal Spermatogenesis and Fertility 
Molecular and Cellular Biology  2005;25(15):6346-6354.
Activation of ubiquitination occurs during spermatogenesis and is dependent on the induction of isoforms of the UBC4 family of ubiquitin-conjugating enzymes. The UBC4-testis isoform is testis specific, is induced in round spermatids, and demonstrates biochemical functions distinct from a ubiquitously expressed isoform UBC4-1. To explore further the function of UBC4-testis, mice bearing inactivation of this gene were produced. Homozygous (−/−) mice showed normal body growth and fertility. Although testis weight and morphology were normal in testes from adult mice, examination of young mice during the first wave of spermatogenesis revealed that testes were ∼10% smaller in weight at 40 and 45 days of age but had become normal at 65 days of age. Overall protein content, levels of ubiquitinated proteins, and ubiquitin-conjugating activity did not differ between wild-type and homozygous (−/−) mice. Spermatid number, as well as the motility of spermatozoa isolated from the epididymis, was also normal in homozygous (−/−) mice. To determine whether the germ cells lacking UBC4-testis might be more sensitive to stress, testes from wild-type and knockout mice were exposed to heat stress by implantation in the abdominal cavity. Testes from both strains of mice showed similar rates of degeneration in response to heat. The lack of an obvious phenotype did not appear to be due to induction of other UBC4 isoforms, as shown by two-dimensional gel immunoblotting. Our data indicate that UBC4-testis plays a role in early maturation of the testis and suggest that the many UBC4 isoforms have mixed redundant and specific functions.
doi:10.1128/MCB.25.15.6346-6354.2005
PMCID: PMC1190331  PMID: 16024774
23.  Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference 
To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.
doi:10.1251/bpo101
PMCID: PMC545973  PMID: 15682222
Sortilin; Sphingolipid Activator Proteins
25.  Divergent N-Terminal Sequences Target an Inducible Testis Deubiquitinating Enzyme to Distinct Subcellular Structures 
Molecular and Cellular Biology  2000;20(17):6568-6578.
Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-γ-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action.
PMCID: PMC86134  PMID: 10938131

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