Gas production by Paucilactobacillus wasatchensis WDCO4 is increased in Cheddar cheese containing sodium gluconate.

Paucilactobacillus wasatchensis can use gluconate (GLCN) as well as galactose as an energy source and because sodium GLCN can be added during salting of Cheddar cheese to reduce calcium lactate crystal formation, our primary objective was to determine if the presence of GLCN in cheese is another risk factor for unwanted gas production leading to slits in cheese. A secondary objective was to calculate the amount of CO2 produced during storage and to relate this to the amount of gas-forming substrate that was utilized. Ribose was added to promote growth of Pa. wasatchensis WDC04 (P.waWDC04) to high numbers during storage. Cheddar cheese was made with lactococcal starter culture with addition of P.waWDC04 on 3 separate occasions. After milling, the curd was divided into six 10-kg portions. To the curd was added (A) salt, or salt plus (B) 0.5% galactose + 0.5% ribose (similar to previous studies), (C) 1% sodium GLCN, (D) 1% sodium GLCN + 0.5% ribose, (E) 2% sodium GLCN, (F) 2% sodium GLCN + 0.5% ribose. A vat of cheese without added P.waWDC04 was made using the same milk and a block of cheese used as an additional control. Cheeses were cut into 900-g pieces, vacuum packaged and stored at 12°C for 16 wk. Each month the bags were examined for gas production and cheese sampled and tested for lactose, galactose and GLCN content, and microbial numbers. In the control cheese, P.waWDC04 remained undetected (i.e., <104 cfu/g), whereas in cheeses A, C, and E it increased to 107 cfu/g, and when ribose was included with salting (cheeses B, D, and F) increased to 108 cfu/g. The amount of gas (measured as headspace height or calculated as mmoles of CO2) during 16 wk storage was increased by adding P.waWDC04 into the milk, and by adding galactose or GLCN to the curd. Galactose levels in cheese B were depleted by 12 wk while no other cheeses had residual galactose. Except for cheese D, the other cheeses with GLCN added (C, E and F) showed little decline in GLCN levels until wk 12, even though gas was being produced starting at wk 4. Based on calculations of CO2 in headspace plus CO2 dissolved in cheese, galactose and GLCN added to cheese curd only accounted for about half of total gas production. It is proposed that CO2 was also produced by decarboxylation of amino acids. Although P.waWDC04 does not have all the genes for complete conversion and decarboxylation of the amino acids in cheese, this can be achieved in conjunction with starter culture lactococcal. Adding GLCN to curd can now be considered another confirmed risk factor for unwanted gas production during storage of Cheddar cheese that can lead to slits and cracks in cheese. Putative risk factors now include having a community of bacteria in cheese leading to decarboxylation of amino acids and release of CO2 as well autolysis of the starter culture that would provide a supply of ribose that can promote growth of Pa. wasatchensis.

The effect of air temperature, velocity and humidity on respiration rate and rectal temperature as an expression of heat stress in gestating sows.

Global warming combined with increased production (i.e. more piglets, more milk and consequently more heat) means that sows are more often challenged by heat stress. The objective was to develop an effective temperature (ET) equation to predict how air temperature, velocity and humidity affect the respiration rate (RR), rectal temperature (RT) and skin temperature (ST) as an expression of heat stress in gestating sows in order to elucidate the relationship between the thermal parameters and the sows' perception of the environment. The experimental room was equipped with a negative pressure ventilation system with diffuse air inlet through the ceiling, electrical heaters, steam generators and dehumidifiers. An air distribution unit was constructed to generate vertical air velocity. A total of 16 gestating sows were exposed to three temperatures (25°C, 29°C and 33°C), two levels of relative humidity (30% and 70%) and three levels of air velocity (0.2 ms-1, 1 ms-1 and 2.5 ms-1). The RR, RT and ST were recorded every 30 min throughout the three 2-h test periods. The estimated effects of humidity and velocity in relation to effect of temperature was nearly independent of whether it was determined from RR or RT, whereas the effect of humidity was much smaller when determined from ST. High coefficients of determination (>0.97) were found for the second order relationship between the estimated ET and RR, RT and ST. An increase in relative humidity from 50 to 70% corresponded to an increase in ET of 0.9°C, while an increase in air velocity from 0.2 to 1.0 ms-1 corresponded to a decrease in ET of 1.2°C. The applied ET equation was useful for expressing the combined effect of temperature, humidity and velocity on animals exposed to heat stress. However, multiplying the effect of velocity by the temperature gradient between the animal and the surrounding air did not improve the estimation.

Voice break in boys-temporal relations with other pubertal milestones and likely causal effects of BMI.

STUDY QUESTION: How is timing of voice break related to other male pubertal milestones as well as to BMI? SUMMARY ANSWER: We provide a comprehensive temporal analysis of male pubertal milestones, including reproductive hormone dynamics, confirm voice break as a late milestone of male puberty and report a likely causal relationship between higher BMI and earlier age at voice break in men. WHAT IS KNOWN ALREADY: Voice break represents a late pubertal milestone and recalled age at voice break is frequently used in epidemiological studies as a measure of puberty. In contrast, clinical studies use mainly testicular enlargement and/or genital tanner stage as the marker of pubertal onset. However, neither correlation of pubertal milestones nor reproductive hormone dynamics have been assessed in detail previously. Further, although BMI and puberty timing are known to be closely linked, cause and effect between these traits are not known. STUDY DESIGN, SIZE, DURATION: The study included a population-based mixed cross-sectional and longitudinal cohort (2006-2014, COPENHAGEN Puberty Study) of 730 healthy Danish boys. Data for 55 871 male research participants from the 23andMe study were obtained, including genome-wide single nucleotide polymorphism data and age at voice break. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed a detailed evaluation of pubertal milestones and reproductive hormone levels (study population 1). A Mendelian randomization (MR) approach was used to determine the likely causal link between BMI and timing of voice break (study population 2). MAIN RESULTS AND THE ROLE OF CHANCE: Voice break occurred at mean age 13.6 (95% CI: 13.5-13.8) years. At voice break, mean (95% CI) testosterone levels, LH levels and bi-testicular volume were 10.9 (10.0-11.7) nmol/L, 2.4 (2.2-2.5) IU/L and 24 (23-25) mL, respectively. Voice break correlated moderately strongly with timing of male pubertal milestones, including testicular enlargement, gonadarche, pubarche, sweat odor, axillary hair growth and testosterone above limit of detection (r2 range: 0.43-0.61). Timing of all milestones was negatively associated with age-specific BMI (all P ≤ 0.001). MR analyses inferred likely causal effects of higher BMI on earlier voice break in males (-0.35 years/approximate SD, P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Participation rate of the population-based cohort was 25%. Further, boys that were followed longitudinally were examined approximately every 6 months limiting the time resolution of pubertal milestones. Using adult BMI as exposure instead of prepubertal BMI in the MR analysis and the known inaccuracies of the testosterone immunoassay at low testosterone levels may be further limitations. WIDER IMPLICATIONS OF THE FINDINGS: We provide valuable normative data on the temporal relation of male pubertal milestones. Further, the likely causal relationship between BMI and puberty timing highlights the importance of preventing obesity in childhood. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Danish Agency for Science, Technology and Innovation (09-067 180); Danish Ministry of the Environment, CeHoS (MST-621-00 065); Capital Region of Denmark (R129-A3966); Ministry of Higher Education and Science (DFF-1331-00 113); Innovation Fund Denmark (InnovationsFonden, 14-2013-4); The International Center for Research and Research Training in Endocrine Disrupting Effects of Male Reproduction and Child Health. B.H., F.R.D., J.R.B.P. and K.K.O. are supported by the Medical Research Council (MC_UU_12015/2). The 23andMe study is supported by the National Human Genome Research Institute of the National Institutes of Health (R44HG006981). Members of the 23andMe Research Team are employees of 23andMe, Inc. and hold stock or stock options in 23andMe. TRIAL REGISTRATION NUMBER: NCT01411527.

Survey on the CRISPR arrays in Lactobacillus helveticus genomes.

Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacteria that is mainly used in the manufacture of Swiss type and long-ripened Italian hard cheeses. In this study, the presence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were analysed in 25 L. helveticus genomes and identified in 23 of these genomes. A total of 40 CRISPR loci were identified and classified into five main families based on CRISPR repeats: Ldbu1, Lsal1, Lhel1, Lhel2 and a new repeat family named Lhel3. Spacers had a size between 30 and 40 bp whereas repeats have an average size of 30 bp, with three longer repeats. The analysis displayed the presence of conserved spacers in 23 of the 40 CRISPR loci. A geographical distribution of L. helveticus isolates with similar CRISPR spacer array profiles were not observed. Based on the presence of the signature protein Cas3, all CRISPR loci belonged to Type I. This analysis demonstrated a great CRISPR array variability within L. helveticus, which could be a useful tool for genotypic strain differentiation. A next step will be to understand the possible role of CRISPR/Cas system for the resistance of L. helveticus to phage infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus helveticus, a lactic acid bacteria species widely used as starter culture in the dairy industry has recently also gained importance as health-promoting culture in probiotic and nutraceutical food products. The CRISPR/Cas system, a well-known molecular mechanism that provides adaptive immunity against exogenous genetic elements such as bacteriophages and plasmids in bacteria, was recently found in this species. In this study, we investigated the presence and genetic heterogeneity of CRISPR loci in 25 L. helveticus genomes. The results presented here represent an important step on the way to manage phage resistance, plasmid uptake and genome editing in this species.

Training and validation of a novel 4-miRNA ratio model (MiCaP) for prediction of postoperative outcome in prostate cancer patients.

Background: New molecular biomarkers for prostate cancer (PC) prognosis are urgently needed. Ratio-based models are attractive, as they require no additional normalization. Here, we train and independently validate a novel 4-miRNA prognostic ratio model for PC. Patients and methods: By genome-wide miRNA expression profiling of PC tissue samples from 123 men who underwent radical prostatectomy (RP) (PCA123, training cohort), we identified six top candidate prognostic miRNAs and systematically tested their ability to predict postoperative biochemical recurrence (BCR). The best miRNA-based prognostic ratio model (MiCaP) was validated in two independent cohorts (PCA352 and PCA476) including >800 RP patients in total. Clinical end points were BCR and prostate cancer-specific survival (CSS). The prognostic potential of MiCaP was assessed by univariate and multivariate Cox-regression analyses and Kaplan-Meier analyses. Results: We identified a 4-miRNA ratio model, MiCaP (miR-23a-3p×miR-10b-5p)/(miR-133a×miR-374b-5p), that predicted time to BCR independently of routine clinicopathologic variables in the training cohort (PCA123) and was successfully validated in two independent RP cohorts. In addition, MiCaP was a significant predictor of CSS in univariate analysis [HR 3.35 (95% CI 1.34 - 8.35), P = 0.0096] and in multivariate analysis [HR 2.43 (95% CI 1.45-4.07), P = 0.0210]. As proof-of-principle, we also analyzed MiCaP in plasma samples from 111 RP patients. A high MiCaP score in plasma was significantly associated with BCR (P = 0.0036, Kaplan-Meier analysis). Limitations include low mortality rates (CSS: 5.4%). Conclusions: We identified a novel 4-miRNA ratio model (MiCaP) with significant independent prognostic value in three RP cohorts, indicating promising potential to improve PC risk stratification.

Clinical diagnosis of Larsen syndrome, Stickler syndrome and Loeys-Dietz syndrome in a 19-year old male: a case report.

BACKGROUND: Larsen syndrome is a hereditary disorder characterized by osteochondrodysplasia, congenital large-joint dislocations, and craniofacial abnormalities. The autosomal dominant type is caused by mutations in the gene that encodes the connective tissue protein, filamin B (FLNB). Loeys-Dietz syndrome (LDS) is an autosomal dominant connective tissue disorder characterized by arterial aneurysms, dissections and tortuosity, and skeletal, including craniofacial, manifestations. Mutations in five genes involved in the transforming growth factor beta (TGF-ß) signaling pathway cause five types of LDS. Stickler syndrome is a genetically heterogeneous arthro-ophthalmopathy caused by defects in collagen, exhibiting a wide specter of manifestations in connective tissue. A rare case is reported that was diagnosed with all these three hereditary connective tissue disorders. CASE PRESENTATION: A 19 year-old, Norwegian male with a clinical diagnosis of Larsen syndrome and with healthy, non-consanguineous parents attended a reference center for rare connective tissue disorders. Findings at birth were hypotonia, joint hypermobility, hyperextended knees, adductovarus of the feet, cervical kyphosis, craniofacial abnormalities, and an umbilical hernia. From toddlerhood, he required a hearing aid due to combined conductive and sensorineural hearing loss. Eye examination revealed hyperopia, astigmatism, and exotropia. At 10 years of age, he underwent emergency surgery for rupture of an ascending aortic aneurysm. At 19 years of age, a diagnostic re-evaluation was prompted by the findings of more distal aortic dilation, tortuosity of precerebral arteries, and skeletal findings. High throughput sequencing of 34 genes for hereditary connective tissue disorders did not identify any mutation in FLNB, but did identify a de novo missense mutation in TGFBR2 and a nonsense mutation in COL2A1 that was also present in his unaffected father. The diagnosis was revised to LDS Type 2. The patient also fulfills the proposed criteria for Stickler syndrome with bifid uvula, hearing loss, and a known mutation in COL2A1. CONCLUSION: LDS should be considered in patients with a clinical diagnosis of Larsen syndrome, in particular in the presence of arterial aneurysms or tortuosity. Due to genetic heterogeneity and extensive overlap of clinical manifestations, genetic high throughput sequencing analysis is particularly useful for the differential diagnosis of hereditary connective tissue disorders.

The influence of blood pressure management on neurological outcome in endovascular therapy for acute ischaemic stroke.

BACKGROUND: Observational studies have suggested that low blood pressure and blood pressure variability may partially explain adverse neurological outcome after endovascular therapy with general anaesthesia (GA) for acute ischaemic stroke. The aim of this study was to further examine whether blood pressure related parameters during endovascular therapy are associated with neurological outcome. METHODS: The GOLIATH trial randomised 128 patients to either GA or conscious sedation for endovascular therapy in acute ischaemic stroke. The primary outcome was 90 day modified Rankin Score. The haemodynamic protocol aimed at keeping the systolic blood pressure >140 mm Hg and mean blood pressure >70 mm Hg during the procedure. Blood pressure related parameters of interest included 20% reduction in mean blood pressure; mean blood pressure <70 mm Hg, <80 mm Hg, and <90 mm Hg, respectively; time with systolic blood pressure <140 mm Hg; procedural minimum and maximum mean and systolic blood pressure; mean blood pressure at the time of groin puncture; postreperfusion mean blood pressure; blood pressure variability; and use of vasopressors. Sensitivity analyses were performed in the subgroup of reperfused patients. RESULTS: Procedural average mean and systolic blood pressures were higher in the conscious sedation group (P<0.001). The number of patients with mean blood pressure <70-90 mm Hg and systolic blood pressure <140 mm Hg, blood pressure variability, and use of vasopressors were all higher in the GA group (P<0.001). There was no statistically significant association between any of the examined blood pressure related parameters and the modified Rankin Score in the overall patient population, and in the subgroup of patients with full reperfusion. CONCLUSION: We found no statistically significant association between blood pressure related parameters during endovascular therapy and neurological outcome. CLINICAL TRIAL REGISTRATION: NCT 02317237.

Accelerating atomic structure search with cluster regularization.

We present a method for accelerating the global structure optimization of atomic compounds. The method is demonstrated to speed up the finding of the anatase TiO2(001)-(1 × 4) surface reconstruction within a density functional tight-binding theory framework using an evolutionary algorithm. As a key element of the method, we use unsupervised machine learning techniques to categorize atoms present in a diverse set of partially disordered surface structures into clusters of atoms having similar local atomic environments. Analysis of more than 1000 different structures shows that the total energy of the structures correlates with the summed distances of the atomic environments to their respective cluster centers in feature space, where the sum runs over all atoms in each structure. Our method is formulated as a gradient based minimization of this summed cluster distance for a given structure and alternates with a standard gradient based energy minimization. While the latter minimization ensures local relaxation within a given energy basin, the former enables escapes from meta-stable basins and hence increases the overall performance of the global optimization.

Cryopreservation of ovarian tissue for fertility preservation in a large cohort of young girls: focus on pubertal development.

STUDY QUESTION: Is there an association between the need for medical puberty induction and the diagnosis or treatment received in girls who have undergone cryopreservation of ovarian tissue for fertility preservation? SUMMARY ANSWER: There was a clear association between the intensity of treatment received and requirement for medical puberty induction but no association with the diagnosis. WHAT IS KNOWN ALREADY: Although it cannot be predicted which girls will become infertile or develop premature ovarian insufficiency (POI) following intensive chemotherapy or irradiation, patients who are at high risk of POI should be offered ovarian tissue cryopreservation (OTC). This includes girls who are planned to receive either high doses of alkylating agents, conditioning regimen before stem cell transplantation (SCT), total body irradiation (TBI) or high radiation doses to the craniospinal, abdominal or pelvic area. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study. In total, 176 Danish girls under 18 years of age have had OTC performed over a period of 15 years. An overview of the girls' diagnoses and mean age at OTC as well as the number of deceased is presented. Of the 176 girls, 38 had died and 46 girls were still younger than 12 years so their pubertal development cannot be evaluated yet. For the 60 girls who had OTC performed after 12 years of age, the incidence of POI was evaluated and in the group of 32 girls who were younger than 12 years at OTC, the association between the diagnosis and received treatment and the requirement for medical puberty induction was examined. PARTICIPANTS/MATERIALS, SETTING, METHODS: The need for medical puberty induction was assessed in 32 girls who were prepubertal at the time of OTC. MAIN RESULTS AND THE ROLE OF CHANCE: Indications for OTC were allogeneic SCT for leukaemia, myelodysplastic syndrome or benign haematological disorders, autologous SCT for lymphoma or sarcoma, and irradiation to the pelvis or to the spinal axis. The mean age at OTC of the 176 girls were 11.3 years. The two most prevalent diagnoses of the 176 girls were malignant tumours and malignant haematological diseases. Among the 32 prepubertal girls, 12 received high dose chemotherapy and either TBI prior to SCT or irradiation to the pelvis, abdomen or the spinal axis, 13 received high dose alkylating agents but no irradiation prior to SCT, six received alkylating agents as part of conventional chemotherapy and one patient had a genetic metabolic disorder and did not receive gonadotoxic treatment. Among these 32 girls, 23 did not undergo puberty spontaneously and thus received medical puberty induction. Among the nine girls, who went through spontaneous puberty, four had received high dose alkylating agents and five had received conventional chemotherapy. LIMITATIONS REASONS FOR CAUTION: All information was retrieved retrospectively from patient records, and thus some information was not available. WIDER IMPLICATIONS OF THE FINDINGS: OTC should be recommended to all young girls, who present a high risk of developing ovarian insufficiency and/or infertility following high dose chemotherapy and/or irradiation. STUDY FUNDING/COMPETING INTERESTS: The Childhood Cancer Foundation (2012-2016) and the EU interregional project ReproHigh are thanked for having funded this study. They had no role in the study design, collection and analysis of the data or writing of the report. The authors have no conflict of interest to disclose.

Editor's Choice - The National Norwegian Carotid Study: Time from Symptom Onset to Surgery is too Long, Resulting in Additional Neurological Events.

OBJECTIVE/BACKGROUND: The objective was to observe for 1 year all patients in Norway operated on for symptomatic carotid stenosis with respect to (i) the time from the index event to surgery and neurological events during this time; (ii) the level in the healthcare system causing delay of surgical treatment; and (iii) the possible relationship between peri-operative use of platelet inhibitors and neurological events while awaiting surgery. METHODS: This was a prospective national multicentre study of a consecutive series of symptomatic patients. Patients were eligible for inclusion when referred for surgery. An index event was defined as the neurological event prompting contact with the healthcare system. All 15 departments in Norway performing carotid endarterectomy (CEA) participated. RESULTS: Three hundred and seventy one patients were eligible for inclusion between 1 April 2014 and 31 March 2015, and 368 patients (99.2%) were included. Fifty-four percent of the patients contacted their general practitioner on the day of the index event. Primary healthcare referred 84.2% of the patients to hospital on the same day as examined. In hospital median time from admission to referral for vascular surgery was 3 days. Median time between referral to the operating unit and actual CEA was 5 days. Overall, 61.7% of the patients were operated on within 2 weeks of the index event. Twelve patients (3.3%) suffered a new neurological event while awaiting surgery. The percentage of patients on dual antiplatelet therapy was lower (25.0%) in this group than among the other patients (62.6%) (p = .008). The combined 30 day mortality and stroke rate was 3.8%. CONCLUSION: This national study with almost complete inclusion and follow-up shows that the delays occur mainly at patient level and in hospital. The delay is associated with new neurological events. Dual antiplatelet therapy is associated with reduced risk of having a new neurological event before surgery.

Comparative assessment of Vibrio virulence in marine fish larvae.

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single-egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty-nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high-virulent strains had acquired virulence genes from other pathogenic Vibrio.

Trends in Abdominal Aortic and Iliac Aneurysm Repairs in Norway from 2001 to 2013.

OBJECTIVE/BACKGROUND: The objective was to examine trends in abdominal aortic and iliac aneurysm repairs in Norway from 2001 to 2013, and study regional variations and organizational developments in this type of vascular surgery. METHODS: This was a retrospective study on aortic and iliac aneurysm repairs using data from the Norwegian Patient Register. The vascular centers were categorized by yearly volume of repairs into small (<18), medium (18-49) and large (≥50). Incidence rates were assessed per 100,000 ≥ 60 years. The percentage of endovascular aneurysm repairs (EVAR) was calculated among the conducted repairs at the three categories of centers and the South-Eastern, Western, Central, and Northern Norway Regional Health Authority (NRHA). RESULTS: The national incidence rates of intact repairs per 100,000 ≥ 60 years increased from 57.4 to 65.7 (p < .01). Ruptured repairs decreased from 19.7 to 9.2 (p < .01). The rate of EVAR increased from 6.0 to 29.9 (p < .01) in intact and from 0.4 to 2.5 (p < .01) in ruptured repairs. The vascular centers were reduced from 25 to 16. The rate of EVAR was 27.1% (p < .01) higher at large centers and 7.9% (p < .03) higher at medium centers compared with small centers, and from 11.1% to 15.7% higher (p < .01) at the Central, Western, and Northern NRHA compared with the South-Eastern NRHA, which had the most centers (also in the large category). The national increase in intact EVAR from 10.6% to 43.3% was less compared with many other Western countries. CONCLUSION: During the study period the rates of intact repairs increased while the ruptured repairs decreased. EVAR was associated with centers performing high volumes of abdominal aortic and iliac aneurysm repairs and regional authorities organized with few centers.

Mutations in the TLR3 signaling pathway and beyond in adult patients with herpes simplex encephalitis.

Herpes simplex encephalitis (HSE) in children has previously been linked to defects in type I interferon production downstream of Toll-like receptor (TLR)3. In the present study, we used whole-exome sequencing to investigate the genetic profile of 16 adult patients with a history of HSE. We identified novel mutations in IRF3, TYK2 and MAVS, molecules involved in generating innate antiviral immune responses, which have not previously been associated with HSE. Moreover, data revealed mutations in TLR3, TRIF, TBK1 and STAT1 known to be associated with HSE in children but not previously described in adults. All discovered mutations were heterozygous missense mutations, the majority of which were associated with significantly decreased antiviral responses to HSV-1 infection and/or the TLR3 agonist poly(I:C) in patient peripheral blood mononuclear cells compared with controls. Altogether, this study demonstrates novel mutations in the TLR3 signaling pathway in molecules previously identified in children, suggesting that impaired innate immunity to HSV-1 may also increase susceptibility to HSE in adults. Importantly, the identification of mutations in innate signaling molecules not directly involved in TLR3 signaling suggests the existence of innate immunodeficiencies predisposing to HSE beyond the TLR3 pathway.

Enhancing the effectiveness of diabetes self-management education: the diabetes literacy project.

Patient empowerment through self-management education is central to improving the quality of diabetes care and preventing Type 2 Diabetes. Although national programs exist, there is no EU-wide strategy for diabetes self-management education, and patients with limited literacy face barriers to effective self-management. The Diabetes Literacy project, initiated with the support of the European Commission, aims to fill this gap. The project investigates the effectiveness of diabetes self-management education, targeting people with or at risk of Type 2 Diabetes in the 28 EU Member States, as part of a comprehensive EU-wide diabetes strategy. National diabetes strategies in the EU, US, Taiwan, and Israel are compared, and diabetes self-management programs inventorized. The costs of the diabetes care pathway are assessed on a per person basis at national level. A comparison is made of the (cost)-effectiveness of different methods for diabetes self-management support, and the moderating role of health literacy, organization of the health services, and implementation fidelity of education programs are considered. Web-based materials are developed and evaluated by randomized trials to evaluate if interactive internet delivery can enhance self-management support for people with lower levels of health literacy. The 3-year project started in December 2012. Several literature reviews have been produced and protocol development and research design are in the final stages. Primary and secondary data collection and analysis take place in 2014. The results will inform policy decisions on improving the prevention, treatment, and care for persons with diabetes across literacy levels.

Socio-economic correlates of functional health literacy among patients of primary health care in Kosovo.

OBJECTIVES: Functional health literacy (FHL) has been related to individual characteristics, ill-health and disease knowledge. However, the information about FHL in Kosovo is very limited and thus the aim of this study was to assess the demographic and socio-economic correlates of FHL among users of primary health care in Kosovo, a postconflict country in the Western Balkans. STUDY DESIGN: Cross-sectional study. METHODS: A cross-sectional study was conducted in Kosovo between November 2012-February 2013, including a representative sample of 1035 consecutive primary care users aged ≥18 years (60% females; overall mean age: 44.3 ± 16.9 years; overall response rate: 86%). Test of Functional Health Literacy in Adults (TOFHLA) was used to assess FHL. General linear model and logistic regression were used to assess the association of TOFHLA score with demographic and socio-economic characteristics. RESULTS: Overall, four out of five participants exhibited inadequate or marginal FHL in this Kosovo sample. FHL score was independently and inversely related to age, but positively associated with educational attainment and being in a situation other than unemployed. CONCLUSIONS: Limited or marginal FHL was very common among primary care users in Kosovo and considerably higher than in the neighbouring Serbia. The low health literacy levels in Kosovo may provide an additional barrier towards achievement of health care goals. There is a need to design and implement suitable and effective educational and health system interventions in the Kosovo context.

Prognostic significance of aberrantly silenced ANPEP expression in prostate cancer.

BACKGROUND: Novel biomarkers for prostate cancer (PC) are urgently needed. This study investigates the expression, epigenetic regulation, and prognostic potential of ANPEP in PC. METHODS: Aminopeptidase N (APN; encoded by ANPEP) expression was analysed by immunohistochemistry using tissue microarrays representing 267 radical prostatectomy (RP) and 111 conservatively treated (CT) PC patients. Clinical end points were recurrence-free survival (RFS) and cancer-specific survival (CSS), respectively. The ANPEP promoter methylation levels were determined by bisulphite sequencing or MethyLight analysis in 278 nonmalignant and PC tissue samples, and in cell lines. RESULTS: The APN expression was significantly downregulated in PC compared with nonmalignant prostate tissue samples. Aberrant promoter hypermethylation was frequently observed in PC tissue samples, and 5-aza-2'-deoxycytidine induced ANPEP expression in three hypermethylated prostate cell lines, suggesting epigenetic silencing. Negative APN immunoreactivity was significantly associated with short RFS and short CSS in the RP and CT cohort, respectively, independently of routine clinicopathological predictors. Combining APN with a known angiogenesis marker (vascular endothelial growth factor or microvessel density) improved risk prediction significantly in both cohorts. CONCLUSION: Our results suggest negative APN immunoreactivity as a new independent adverse prognostic factor for patients with clinically localised PC and, furthermore, that epigenetic mechanisms are involved in silencing of ANPEP in PC.

Endothelial dysfunction, increased inflammation, and activated coagulation in HIV-infected patients improve after initiation of highly active antiretroviral therapy.

OBJECTIVES: Endothelial dysfunction and inflammation have been demonstrated to be markers of cardiovascular risk. We investigated the effects of HIV infection per se and the antiretroviral treatment prescribed on the levels of risk factors of cardiovascular disease. METHODS: This was a prospective study of 20 treatment-naïve, nonsmoking, HIV-positive patients examined before and after 3 months of treatment with a protease inhibitor (PI)-containing regimen followed by 3 months of treatment with nonnucleoside reverse transcriptase inhibitor (NNRTI)-containing therapy. Parameters of inflammation, endothelial function and coagulation were examined. The results were compared with those for an age- and gender-matched, nonsmoking, healthy control group. RESULTS: Compared with controls, treatment-naïve HIV-infected patients exhibited endothelial dysfunction [flow-mediated dilation (FMD) 108 vs. 111% for HIV-infected vs. control groups, respectively; P < 0.05] and activation [von Willebrand factor 2.0 vs. 0.9 U/l; soluble intercellular adhesion molecule (sICAM) 313 vs. 211 ng/L, respectively; P < 0.01]. Inflammation [C-reactive protein (CRP) 24 vs. 8.6 nmol/L; fibrinogen 9.4 vs. 8.6 µmol/L, respectively; P < 0.05] and coagulation/fibrinolysis (D-dimers 0.55 vs. 0.23 µg/mL, respectively; P < 0.01) were increased. Initiating therapy resulted in normalization of FMD and a significant decrease in endothelial activation and CRP. CONCLUSION: Endothelial dysfunction together with increased inflammation and coagulation were more prevalent in untreated HIV-infected patients compared with controls. These cardiovascular risk factors improved with treatment, although not all parameters normalized after 6 months.

Protein expression studies of desmoplakin mutations in cardiomyopathy patients reveal different molecular disease mechanisms.

Mutations in the gene for desmoplakin (DSP) may cause arrhythmogenic right ventricular cardiomyopathy (ARVC) and Carvajal syndrome (CS). Desmoplakin is part of all desmosomes, which are abundantly expressed in both myocardial and epidermal tissue and serve as intercellular mechanical junctions. This study aimed to investigate protein expression in myocardial and epidermal tissue of ARVC and CS patients carrying DSP mutations in order to elucidate potential molecular disease mechanisms. Genetic investigations identified three ARVC patients carrying different heterozygous DSP mutations in addition to a homozygous DSP mutation in a CS patient. The protein expression of DSP in mutation carriers was evaluated in biopsies from myocardial and epidermal tissue by immunohistochemistry. Keratinocyte cultures were established from skin biopsies of mutation carriers and characterized by reverse transcriptase polymerase chain reaction, western blotting, and protein mass spectrometry. The results showed that the mutation carriers had abnormal DSP expression in both myocardial and epidermal tissue. The investigations revealed that the disease mechanisms varied accordingly to the specific types of DSP mutation identified and included haploinsufficiency, dominant-negative effects, or a combination hereof. Furthermore, the results suggest that the keratinocytes cultured from patients are a valuable and easily accessible resource to elucidate the effects of desmosomal gene mutations in humans.

Thromboembolic complications in Fontan patients: population-based prevalence and exploration of the etiology.

After the Fontan procedure, patients face an increased risk for thromboembolic events (TE). The etiology for this increased thrombogenecity is incompletely understood. This study aimed to determine the prevalence of TE in Danish Fontan patients and to bring new insights into the etiology of TE. Using a population-based design, we retrospectively identified all TEs in 210 Fontan patients. Whole blood assays (thromboelastography, thromboelastography functional fibrinogen and Multiplate) reflecting global hemostasis, clot strength and platelet aggregation were analyzed prospectively in 112 patients and plasma was analyzed in 76 patients for biomarkers reflecting endothelial-, glycocalyx-, platelet-, and fibrinolysis function (histone-complexed DNA fragments, Protein C, soluble CD40 ligand, soluble thrombomodulin, syndecan-1, tissue-type plasminogen activator). The results were compared in groups stratified according to age, antithrombotic therapy, TE, and glycocalyx degradation (syndecan-1 < or ≥ median). Correlation between biomarkers and demographic-, anatomical-, clinical- and biochemical parameters was investigated. The prevalence of TE was 8.1 % after a mean follow-up of 8.4 years. None of the stratified groups demonstrated evidence of hypercoagulability in the whole blood assays and no unexpected significant differences were found between the groups. All biomarkers, except protein C, correlated with one another and after stratification of glycocalyx degradation only syndecan-1 levels ≥ median correlated with other biomarkers. The prevalence of TEs was 8.1 % after mean follow-up of 8.4 years. Overall, the hemostatic profile appeared normal, however, in a subset of patients, evidence of some endothelial activation/damage including glycocalyx degradation and fibrinolysis was found, identifying a potentially more thrombogenic group.

The implementation and evaluation of a mentoring program for Bachelor of Midwifery students in the clinical practice environment.

AIM: To describe the implementation and evaluation of a midwife/midwifery student-mentoring program in one Local Health District in Sydney NSW Australia. BACKGROUND: Evidence suggests well designed and supported midwife/midwifery student mentorship programs can make a difference to the clinical placement experiences and attrition rates of midwifery students. DESIGN: In the evaluation of the mentoring program, we used surveys, focus groups and individual interviews. METHODS: Eighty-six participants, including midwife mentors, midwifery students, non-mentor midwives and midwifery managers participated in the evaluation. Quantitative data were analysed using descriptive statistics and qualitative data, content analysis. RESULTS: The mentoring program increased midwives' mentoring skills and was beneficial to their professional growth and leadership skills. Students reported positive outcomes including someone to talk to, emotional support and a sense of belonging. Mentoring programs require structure, mentor training, organisational support and transparency. CONCLUSION: The mentoring program provided benefits to both midwifery mentors and students and demonstrated the value of a structured and supported mentoring program for midwifery students.