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PurposeObstructive sleep apnea (OSA) is a common, prevalent, and underdiagnosed disorder. Its lack of diagnosis and treatment is associated with increased morbidity and mortality. Previous screening questionnaires investigated parameters including body mass index, age, neck circumference, and sex, in addition to symptoms. This study aimed to validate a new Italian, self-administered, and easy-to-use six-item questionnaire that evaluates only subjective symptoms.Patients and methodsThe present study included 2622 patients (male, 2011; female, 611). Patients who were at least 18 years old, spoke Italian, referred to our sleep clinic for possible OSA, and completed the self-administered SANReSP questionnaire were recruited for the study. The predictive performance of the questionnaire was also evaluated.ResultsNocturnal study showed 89.9% of OSA patients had apnea-hypopnea index (AHI) ≥ 5/h; 68.7%, AHI ≥ 15/h; and 48.2%, AHI ≥ 30/h. The optimal SANReSP score for AHI ≥ 5/h was >3 with a sensitivity and specificity of 74.76% and 67.92%, respectively, and an area under receiver operating characteristic curve (ROC) of 0.76. For moderate-severe OSA, the optimal SANReSP score was >3 (sensitivity, 78.18%; specificity, 46.53%; ROC, 0.66). For severe OSA, the optimal SANReSP score was >4 (sensitivity, 59.10%; specificity, 64.73%; ROC, 0.65). The probability of OSA increased with higher SANReSP scores (98.7% and 97.9% in men and women, respectively).ConclusionThe SANReSP questionnaire is a short, easy-to-use, and self-administered screening tool for OSA. Its performance is similar to that of other widely used questionnaires; furthermore, it is advantageous in that it does not require anthropometric measurements.

Type 1 diabetes and thyroid disease are T cell-dependent autoimmune endocrinopathies. The standard substitutive administration of the deficient hormones does not halt the autoimmune process; therefore, development of immunotherapies aiming to preserve the residual hormonal cells, is of crucial importance. PTPN22 C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant, plays a potential pathophysiological role in autoimmunity. The PTPN22 encoded protein Lyp is a negative regulator of T cell antigen receptor signaling; R620W variant, leading to a gain of function with paradoxical reduced T cell activation, may represent a valid therapeutic target. We aimed to develop novel wild type PTPN22 short interfering RNA duplexes (siRNA) and optimize their delivery into Jurkat T cells and PBMC by using liposomal carriers. Conformational stability, size and polydispersion of siRNA in lipoplexes was measured by CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation was assessed by confocal microscopy and flow cytometry analysis. Their effect on Lyp expression was evaluated by means of Western Blot and confocal microscopy. Functional assays through engagement of TCR signaling were established to evaluate biological consequences of down-modulation. Both Jurkat T cells and PBMC were efficiently transfected by stable custom lipoplexes. Jurkat T cell morphology and proliferation was not affected. Lipoplexes incorporation was visualized in CD3+ but also in CD3- peripheral blood immunotypes without signs of toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression was demonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment of Lyp inhibitory activity was revealed by increase of IL-2 secretion in culture supernatants of PBMC following anti-CD3/CD28 T cell receptor-driven stimulation. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on the selective inhibition of variant PTPN22 allele using lipoplexes of siRNA antisense oligomers.

We assessed whether obstructive sleep apnea (OSA) and nocturnal hypoxemia are associated with severity of liver fibrosis and carotid atherosclerosis in patients with biopsy-proven NAFLD and low prevalence of morbid obesity. Secondary aim was to explore the association of OSA and hypoxemia with NASH and severity of liver pathological changes.Consecutive patients (n = 126) with chronically elevated ALT and NAFLD underwent STOP-BANG questionnaire to estimate OSA risk and ultrasonographic carotid assessment. In patients accepting to perform cardiorespiratory polygraphy (PG, n = 50), OSA was defined as an apnea/hypopnea index ≥5. A carotid atherosclerotic plaque was defined as a focal thickening >1.3 mm.Prevalence of high OSA risk was similar in patients refusing or accepting PG (76% vs 68%, p = 0.17). Among those accepting PG, overall OSA prevalence was significantly higher in patients with F2-F4 fibrosis compared to those without (72% vs 44%; p = 0.04). Significant fibrosis was independently associated with mean nocturnal oxygen saturation (SaO2)1 (OR 6.30, 95%C.I. 1.02-12.3; p = 0.01).In NAFLD patients with chronically elevated ALT at low prevalence of morbid obesity, OSA was highly prevalent and indexes of SaO2 resulted independently associated with severity of liver fibrosis and carotid atherosclerosis. These data suggest to consider sleep disordered breathing as a potential additional therapeutic target in severe NAFLD patients.

Type 1 diabetes is an autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Among the genetic variants associated with type 1 diabetes, the C1858T (Lyp) polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene alters the function of T cells but also of B cells in innate and adaptive immunity. The Lyp variant was shown to diminish interferon production and responses upon Toll-like receptor stimulation in macrophages and dendritic cells, possibly leading to uncontrolled infections as triggers of the diabetogenic process. The aim of this study was to unravel the yet uncharacterized effects that the variant could exert on the immune and autoimmune responses, particularly regarding the B cell phenotype, in the peripheral blood lymphocytes of diabetic patients and healthy controls in basal conditions and after unmethylated bacterial DNA CpG stimulation. The presence of the Lyp variant resulted in a significant increase in the percentage of transitional B cells in C/T carriers patients and controls compared to C/C patients and controls, in C/T carrier patients compared to C/C controls and in C/T carrier patients compared to C/C patients. A significant reduction in the memory B cells was also observed in the presence of the risk variant. After four days of CpG stimulation, there was a significant increase in the abundance of IgM+ memory B cells in C/T carrier diabetics than in C/C subjects and in the groups of C/T carrier individuals than in C/C individuals. IgM- memory B cells tended to differentiate more precociously into plasma cells than IgM+ memory B cells in heterozygous C/T subjects compared to the C/C subjects. The increased Toll-like receptor response that led to expanded T cell-independent IgM+ memory B cells should be further investigated to determine the putative contribution of innate immune responses in the disease pathogenesis.

BackgroundWomen with peripheral artery disease (PAD) often have atypical symptoms, late hospital presentations, and worse prognosis. Risk factor identification and management are important. We assessed sex differences in associations of risk factors with PAD.Methods500,207 UK Biobank participants (54.5% women, mean age 56.5 years) without prior hospitalisation of PAD at baseline were included. Examined risk factors included blood pressure, smoking, diabetes, lipids, adiposity, history of stroke or myocardial infarction (MI), socioeconomic status, kidney function, C-reactive protein, and alcohol consumption. Poisson and Cox regressions were used to estimate sex-specific incidence of PAD hospitalisation or death, hazard ratios (HRs), and women-to-men ratios of HRs (RHR) with confidence intervals (CIs).ResultsOver a median of 12.6 years, 2658 women and 5002 men had a documented PAD. Age-adjusted incidence rates were higher in men. Most risk factors were associated with a higher risk of PAD in both sexes. Compared with men, women who were smokers or had a history of stroke or MI had a greater excess risk of PAD (relative to those who never smoked or had no history of stroke or MI): RHR 1.18 (95%CI 1.04, 1.34), 1.26 (1.02, 1.55), and 1.50 (1.25, 1.81), respectively. Higher high-density lipoprotein cholesterol (HDL-C) was more strongly associated with a lower risk of PAD in women than men, RHR 0.81 (0.68, 0.96). Compared to HDL-C at 40 to 60 mg/dL, the lowest level of HDL-C (≤40 mg/dL) was related to greater excess risk in women, RHR 1.20 (1.02, 1.41), whereas the highest level of HDL-C (>80 mg/dL) was associated with lower risk of PAD in women, but higher risk in men, RHR 0.50 (0.38, 0.65).ConclusionsWhile the incidence of PAD was higher in men, smoking and a history of stroke or MI were more strongly associated with a higher risk of PAD in women than men. HDL-C was more strongly associated with a lower risk of PAD in women than men.

[This corrects the article DOI: 10.1371/journal.pone.0276115.].

BackgroundThe use of medications during pregnancy is a common event worldwide. Monitoring medicine prescriptions in clinical practice is a necessary step in assessing the impact of therapeutic choices in pregnant women as well as the adherence to clinical guidelines. The aim of this study was to provide prevalence data on medication use before, during and after pregnancy in the Italian population.MethodsA retrospective prevalence study using administrative healthcare databases was conducted. A cohort of 449,012 pregnant women (15-49 years) residing in eight Italian regions (59% of national population), who delivered in 2016-2018, were enrolled. The prevalence of medication use was estimated as the proportion (%) of pregnant women with any prescription.ResultsAbout 73.1% of enrolled women received at least one drug prescription during pregnancy, 57.1% in pre-pregnancy and 59.3% in postpartum period. The prevalence of drug prescriptions increased with maternal age, especially during the 1st trimester of pregnancy. The most prescribed medicine was folic acid (34.6%), followed by progesterone (19%), both concentrated in 1st trimester of pregnancy (29.2% and 14.8%, respectively). Eight of the top 30 most prescribed medications were antibiotics, whose prevalence was higher during 2nd trimester of pregnancy in women ≥ 40 years (21.6%). An increase in prescriptions of anti-hypertensives, antidiabetics, thyroid hormone and heparin preparations was observed during pregnancy; on the contrary, a decrease was found for chronic therapies, such as anti-epileptics or lipid-modifying agents.ConclusionsThis study represents the largest and most representative population-based study illustrating the medication prescription patterns before, during and after pregnancy in Italy. The observed prescriptive trends were comparable to those reported in other European countries. Given the limited information on medication use in Italian pregnant women, the performed analyses provide an updated overview of drug prescribing in this population, which can help to identify critical aspects in clinical practice and to improve the medical care of pregnant and childbearing women in Italy.

Pectus excavatum (PE) is one of the most frequently reported chest deformities. However, limited studies are available with regard to its CT scan findings in cats. In the present research computed tomographic images of the thoraxes of 14 cats diagnosed with PE has been reviewed. This is one of the first studies exploring the use of CT to characterise PE in animals. The aim of this study was to present characteristic CT features of PE in cats. The introduction of new criteria for better assessing thoracic wall deformity-a correction index (CI) and an asymmetry index (AI)-was also proposed. The study revealed a high variety of morphological features of PE in cats. It was demonstrated that among the 14 cats: cranial PE (an atypical location) occurred in seven cats while seven cats had typical (caudal) PE, long PE occurred in five cats, while short PE had nine cats. Of the 14 cats included in the study eight showed symmetric PE, and asymmetric PE was found insix. Thoracic asymmetry was found in six cats. Six cats had sternal torsion. Based on the Vertebral Index moderate or severe PE was revealed in 11 animals. In the group of cats studied the CI ranged from 12.20 to 32.11. The magnitude of AI did not exceed 10% in any of the cats studied. The study confirmed statistically significant differences in the CI values between groups of cats with different degrees of PE severity (p = 0.02). CT examination showed many PE features that have not been discussed so far. The main benefit of CT examination is its ability to reveal asymmetric PE, thoracic asymmetry and sternal torsion. CI and AI provided a clinically useful tool to quantify thoracic wall deformity in order to obtain comparable results between cats with PE.

Otters are semi-aquatic mammals specialized in feeding on aquatic prey. The Eurasian otter Lutra lutra is the most widely distributed otter species. Despite a low degree of genetic variation across its European range, the population from Great Britain exhibits distinct genetic structuring. We examined 43 skulls of adult Eurasian otters belonging to 18 sampling localities and three genetic clusters (Shetlands, Wales and Scotland). For each sample location, information regarding climate was described using bioclimatic variables from WorldClim, and information on otter diet was extracted from the literature. By using photogrammetry, 3D models were obtained for each skull. To explore any evidence of adaptive divergence within these areas we used a three dimensional geometric morphometric approach to test differences in skull size and shape between areas with genetically distinct populations, as well as the influence of diet, isolation by distance and climate. Males were significantly larger in skull size than females across all the three genetic clusters. Skull shape, but not size, appeared to differ significantly among genetic clusters, with otters from Shetland exhibiting wider zygomatic arches and longer snouts compared to otters from Wales, whereas otters from Scotland displayed intermediate traits. A significant relationship could also be found between skull shape variation, diet as well as climate. Specifically, otters feeding on freshwater fish had more slender and short-snouted skulls compared to otters feeding mostly on marine fish. Individuals living along the coast are characterised by a mixed feeding regime based on marine fish and crustaceans and their skull showed an intermediate shape. Coastal and island otters also had larger orbits and eyes more oriented toward the ground, a larger nasal cavity, and a larger distance between postorbital processes and zygomatic arch. These functional traits could also represent an adaptation to favour the duration and depth of diving, while the slender skull of freshwater feeding otters could improve the hydrodynamics.

This registered report article investigates the role of language as a dimension of social categorization. Our critical aim was to investigate whether categorization based on language occurs even when the languages coexist within the same sociolinguistic context, as is the case in bilingual communities. Bilingual individuals of two bilingual communities, the Basque Country (Spain) and Veneto (Italy), were tested using the memory confusion paradigm in a 'Who said what?' task. In the encoding part of the task, participants were presented with different faces together with auditory sentences. Two different languages of the sentences were presented in each study, with half of the faces always associated with one language and the other half with the other language. Spanish and Basque languages were used in Study 1, and Italian and Venetian dialect in Study 2. In the test phase, the auditory sentences were presented again and participants were required to decide which face uttered each sentence. As expected, participants error rates were high. Critically, participants were more likely to confuse faces from the same language category than from the other (different) language category. The results indicate that bilinguals categorize individuals belonging to the same sociolinguistic community based on the language these individuals speak, suggesting that social categorization based on language is an automatic process.

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors.

BackgroundIn 2017 Public Health England and Sport England commissioned a Consultant-led Sport and Exercise Medicine (SEM) pilot to test the feasibility and acceptability of embedding physical activity interventions in secondary care clinical pathways. The aim of this paper is to report qualitative findings exploring the experience of healthcare professionals (HCPs) and patients involved in the Active Hospital pilot.MethodsQualitative data was collected by semi-structured interviews with Active Hospital pilot SEM Consultants, and staff and patients involved in three clinical pathways. Interviews with SEM Consultants explored the experience of developing and implementing the pilot. Interviews with staff and patients explored the experience of delivering and receiving Active Hospital interventions. Data were analysed thematically.ResultsInterviews identified the importance of the Active Hospital pilot being Consultant-led for the following reasons; i) having trusting relationships with decision makers, ii) having sufficient influence to effect change, iii) identifying champions within the system, and iv) being adaptable to change and ensuring the programme fits within the wider strategic frameworks. HCPs emphasised the importance of the Active Hospital interventions fitting easily within existing work practices, the need for staff training and to tailor interventions for individual patient needs. The Active Hospital pilot was well received by patients, however a lack of dedicated resource and capacity to deliver the intervention was highlighted as a challenge by both patients and HCPs.ConclusionThe SEM Consultants' ability to navigate the political climate of a large National Health Service (NHS) Trust with competing agendas and limited resource was valuable. The interventions were well received and a valued addition to usual clinical care. However, implementation and ongoing delivery of the pilot encountered challenges including lack of capacity within the system and delays with recruiting to the delivery teams in each pathway.

The present pre-registration aims to investigate the role of language as a dimension of social categorization. Our critical aim is to investigate whether language can be used as a dimension of social categorization even when the languages coexist within the same sociolinguistic group, as is the case in bilingual communities where two languages are used in daily social interactions. We will use the memory confusion paradigm (also known as the Who said what? task). In the first part of the task, i.e. encoding, participants will be presented with a face (i.e. speaker) and will listen to an auditory sentence. Two languages will be used, with half of the faces always associated with one language and the other half with the other language. In the second phase, i.e. recognition, all the faces will be presented on the screen and participants will decide which face uttered which sentence in the encoding phase. Based on previous literature, we expect that participants will be more likely to confuse faces from within the same language category than from the other language category. Participants will be bilingual individuals of two bilingual communities, the Basque Country (Spain) and Veneto (Italy). The two languages of these communities will be used, Spanish and Basque (Study 1), and Italian and Venetian dialect (Study 2). Furthermore, we will explore whether the amount of daily exposure to the two languages modulates the effect of language as a social categorization cue. This research will allow us to test whether bilingual people use language to categorize individuals belonging to the same sociolinguistic community based on the language these individuals are speaking. Our findings may have relevant political and social implications for linguistic policies in bilingual communities.

AKI is associated with increased risk of death, prolonged length of stay and development of de-novo chronic kidney disease. The aim of our study is the development and validation of prediction models to identify the risk of AKI in ICU patients up to 7 days. We retrospectively recruited 692 consecutive patients admitted to the ICU at San Bortolo Hospital (Vicenza, Italy) from 1 June 2016 to 31 March 2017: 455 patients were treated as the derivation group and 237 as the validation group. Candidate variables were selected based on a literature review and expert opinion. Admission eGFR< 90 ml/min /1.73 mq (OR 2.78; 95% CI 1.78-4.35; p

Health effects of traffic-related air pollution (TRAP) concentrations in densely populated areas are previously described. However, there is still a lack of knowledge of the health effects of moderate TRAP levels. The aim of the current study, a population-based survey including 16 099 adults (response rate 33%), was to assess the relationship between TRAP estimates and respiratory symptoms in an area with modest levels of traffic; Telemark County, Norway. Respondents reported respiratory symptoms the past 12 months and two TRAP exposure estimates: amount of traffic outside their bedroom window and time spent by foot daily along a moderate to heavy traffic road. Females reported on average more symptoms than males. Significant relationships between traffic outside their bedroom window and number of symptoms were only found among females, with the strongest associations among female occasional smokers (incidence rate ratio [IRR], 1.75, 95% confidence interval (CI) [1.16-2.62] for moderate or heavy traffic compared to no traffic). Significant relationship between time spent daily by foot along a moderate to heavy traffic road and number of symptoms was found among male daily smokers (IRR 1.09, 95% CI [1.04-1.15] per hour increase). Associations between traffic outside bedroom window and each respiratory symptom were found. Significant associations were primarily detected among females, both among smokers and non-smokers. Significant associations between time spent by foot daily along a moderate to heavy traffic road (per hour) and nocturnal dyspnoea (odds ratio (OR) 1.20, 95% CI [1.05-1.38]), nocturnal chest tightness (OR 1.13 [1.00-1.28]) and wheezing (OR 1.14 [1.02-1.29]) were found among daily smokers, primarily men. Overall, we found significant associations between self-reported TRAP exposures and respiratory symptoms. Differences between genders and smoking status were identified. The findings indicate an association between TRAP and respiratory symptoms even in populations exposed to modest levels of TRAP.

AIM:To in detail delineate sex differences in non-small cell lung cancer outcome and investigate possible underlying drivers. METHODS:We performed a nationwide, population-based cohort study using data on all incident cases of lung squamous cell carcinoma (n = 10,325) and adenocarcinoma (n = 23,465) recorded in the Swedish Lung Cancer Register in 2002-2016. Flexible parametric models were applied to compute adjusted female-to-male hazard ratios (aHRs) and standardized survival proportions over follow-up including age, calendar year, education, marital status, birth country, health care region, performance status, smoking history, comorbidities, and tumor location in the final model. RESULTS:Women presented with better performance status, were younger, and more often never-smokers. Women with adenocarcinoma also had lower comorbidity burden, less advanced stage, and were more often EGFR positive. Men with adenocarcinoma had a consistently poorer lung cancer-specific survival across stage; HR 0.69; 95% CI 0.63-0.76 (stage IA-IIB) to 0.94; 95% CI 0.88-0.99 (stage IIIB-IV), remaining largely unchanged after adjustments; aHR 0.74; 95% CI 0.66-0.82 to 0.84; 95% CI 0.81-0.87. The same pattern was observed in squamous cell carcinoma, except in stage IIIA disease, where we found no sex differences in survival. CONCLUSIONS:Men with non-small cell lung cancer have a consistently poorer prognosis, even after careful adjustments for a wide range of prognostic factors. While the pattern was similar in both squamous cell and adenocarcinoma, it was larger and more consistent in the latter.

Disturbances in one-carbon metabolism, intracellular reactions involved in nucleotide synthesis and methylation, likely increase the risk of colorectal cancer (CRC). However, results have been inconsistent. To explore whether this inconsistency could be explained by intertumoral heterogeneity, we evaluated a comprehensive panel of one-carbon metabolism biomarkers and some single nucleotide polymorphisms (SNPs) in relation to the risk of molecular subtypes of CRC defined by mutations in the KRAS and BRAF oncogenes. This nested case-control study included 488 CRC cases and 947 matched controls from two population-based cohorts in the Northern Sweden Health and Disease Study. We analyzed 14 biomarkers and 17 SNPs in prediagnostic blood and determined KRAS and BRAF mutation status in tumor tissue. In a multivariate network analysis, no variable displayed a strong association with the risk of specific CRC subtypes. A non-synonymous SNP in the CTH gene, rs1021737, had a stronger association compared with other variables. In subsequent univariate analyses, participants with variant rs1021737 genotype had a decreased risk of KRAS-mutated CRC (OR per allele = 0.72, 95% CI = 0.50, 1.05), and an increased risk of BRAF-mutated CRC (OR per allele = 1.56, 95% CI = 1.07, 2.30), with weak evidence for heterogeneity (Pheterogeneity = 0.01). This subtype-specific SNP association was not replicated in a case-case analysis of 533 CRC cases from The Cancer Genome Atlas (P = 0.85). In conclusion, we found no support for clear subtype-specific roles of one-carbon metabolism biomarkers and SNPs in CRC development, making differences in CRC molecular subtype distributions an unlikely explanation for the varying results on the role of one-carbon metabolism in CRC development across previous studies. Further investigation of the CTH gene in colorectal carcinogenesis with regards to KRAS and BRAF mutations or other molecular characteristics of the tumor may be warranted.

BACKGROUND:Infection with cytomegalovirus (CMV) remains asymptomatic in most immunocompetent hosts, but is the leading cause of congenital viral infection worldwide and can be life-threatening in immunocompromised individuals. We aimed to assess CMV seroprevalence in a nationally representative sample of adults in Germany and to identify sociodemographic factors associated with CMV seropositivity. METHODS:Blood samples from 6552 participants (18-79 years) of the "German National Health Interview and Examination Survey 1998", a population-based sample of the adult population in Germany, were tested for the presence of CMV antibodies using an Ig-multiplex assay. Weighted seroprevalence was calculated and weighted binomial regression was used to identify factors associated with CMV seropositivity. RESULTS:Overall CMV seroprevalence was 56.7% (95%CI: 54.8-58.7%), with a higher seroprevalence in women (62.3%) than in men (51.0%). Seroprevalence increased with age: from 31.8% to 63.7% in men and from 44.1% to 77.6% in women when comparing the 18-29 with the 70-79 year age-group, respectively. CMV seroprevalence in women of childbearing age (18-45 years) was 51.7%. Factors significantly associated with CMV seropositivity were age, country of birth, smoking status, education, living in northern Germany and number of household members. In addition, having attended child care was associated with seropositivity in men, and number of siblings and living in East Germany in women. CONCLUSION:Our results indicate that half the women of childbearing age were susceptible for primary CMV infection during pregnancy. CMV screening during pregnancy and informing seronegative women about CMV risk reduction measures could prevent congenital CMV infections with its serious consequences.

Everyday, we are bombarded with periodic, exogenous appeals and instructions on how to behave. How do these appeals and instructions affect subsequent coordination? Using experimental methods, we investigate how a one-time exogenous instruction affects subsequent coordination among individuals in a lab. Participants play a minimum effort game repeated 5 times under fixed matching with a one-time behavioral instruction in either the first or second round. Since coordination behavior may vary across countries, we run experiments in Denmark, Spain and Ghana, and map cross-country rankings in coordination with known national measures of fractualization, uncertainty avoidance and long-term orientation. Our results show that exogenous interventions increase subsequent coordination, with earlier interventions yielding better coordination than later interventions. We also find that cross-country rankings in coordination map with published national measures of fractualization, uncertainty avoidance, and long-term orientation.

In the case of the pests inhabiting metal polluted or fields where the use of pesticides is common, a natural selection of resistant individuals can occur. This may pose serious problems for humans, agriculture, as well as the economies of many countries. In this study, the hypothesis that multigenerational (120 generations) exposure to cadmium of a beet armyworm population could be a selecting factor toward a more efficient DNA protection was verified. The hemocytes of individuals from two culture strains (control and Cd-exposed) were treated with H2O2 (a DNA-damaging agent) or PBS (reference). The level of DNA damage was assessed using the Comet assay immediately and 5, 15 and 30 min. after the treatment. The immediate result of the contact with H2O2 was that the level of DNA damage in the hemocytes of the insects from both strains increased significantly. However, in the cells of the Cd-exposed individuals, the level of DNA damage decreased over time, while in the cells from the control insects it remained at the same level with no evidence of repair. These results suggest that efficient defense mechanisms may exist in the cells of insects that have prolonged contact with cadmium. Some evolutionary and trade-off aspects of the phenomenon are discussed. In a wider context, comparing the results obtained in the laboratory with field studies may be beneficial for understanding basic mechanisms of the resistance of an organism. To summarize, the high potential for the repair of DNA damage that was observed in the insects from the cadmium strain may confirm the hypothesis that multigenerational exposure to that metal may possibly contribute to the selection of insects that have a wider tolerance to oxidative stress. However, our investigations of polymorphism using AFLP did not reveal differences between the two main insect strains.

Feeding adaptation, social behaviour, and interspecific interactions related to sexual dimorphism and allometric growth are particularly challenging to be investigated in the high sexual monomorphic Delphinidae. We used geometric morphometrics to extensively explore sexual dimorphism and ontogenetic allometry of different projections of the skull and the mandible of the bottlenose dolphin Tursiops truncatus. Two-dimensional landmarks were recorded on the dorsal, ventral, lateral, and occipital views of the skull, and on the lateral view of the left and the right mandible of 104 specimens from the Mediterranean and the North Seas, differing environmental condition and degree of interspecific associations. Landmark configurations were transformed, standardized and superimposed through a Generalized Procrustes Analysis. Size and shape differences between adult males and females were respectively evaluated through ANOVA on centroid size, Procrustes ANOVA on Procrustes distances, and MANOVA on Procrustes coordinates. Ontogenetic allometry was investigated by multivariate regression of shape coordinates on centroid size in the largest homogenous sample from the North Sea. Results evidenced sexual dimorphic asymmetric traits only detected in the adults of the North Sea bottlenose dolphins living in monospecific associations, with females bearing a marked incision of the cavity hosting the left tympanic bulla. These differences were related to a more refined echolocalization system that likely enhances the exploitation of local resources by philopatric females. Distinct shape in immature versus mature stages and asymmetric changes in postnatal allometry of dorsal and occipital traits, suggest that differences between males and females are established early during growth. Allometric growth trajectories differed between males and females for the ventral view of the skull. Allometric trajectories differed among projections of skull and mandible, and were related to dietary shifts experienced by subadults and adults.

The ability of the Blood Brain Barrier (BBB) to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS) activity and regulating leukocytes' access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS): fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF) that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

We have assembled a network of cell-fate determining transcription factors that play a key role in the specification of the ventral neuronal subtypes of the spinal cord on the basis of published transcriptional interactions. Asynchronous Boolean modelling of the network was used to compare simulation results with reported experimental observations. Such comparison highlighted the need to include additional regulatory connections in order to obtain the fixed point attractors of the model associated with the five known progenitor cell types located in the ventral spinal cord. The revised gene regulatory network reproduced previously observed cell state switches between progenitor cells observed in knock-out animal models or in experiments where the transcription factors were overexpressed. Furthermore the network predicted the inhibition of Irx3 by Nkx2.2 and this prediction was tested experimentally. Our results provide evidence for the existence of an as yet undescribed inhibitory connection which could potentially have significance beyond the ventral spinal cord. The work presented in this paper demonstrates the strength of Boolean modelling for identifying gene regulatory networks.

Complex human diseases can show significant heterogeneity between patients with the same phenotypic disorder. An outlier detection strategy was developed to identify variants at the level of gene transcription that are of potential biological and phenotypic importance. Here we describe a graphical software package (z-score outlier detection (ZODET)) that enables identification and visualisation of gross abnormalities in gene expression (outliers) in individuals, using whole genome microarray data. Mean and standard deviation of expression in a healthy control cohort is used to detect both over and under-expressed probes in individual test subjects. We compared the potential of ZODET to detect outlier genes in gene expression datasets with a previously described statistical method, gene tissue index (GTI), using a simulated expression dataset and a publicly available monocyte-derived macrophage microarray dataset. Taken together, these results support ZODET as a novel approach to identify outlier genes of potential pathogenic relevance in complex human diseases. The algorithm is implemented using R packages and Java.The software is freely available from http://www.ucl.ac.uk/medicine/molecular-medicine/publications/microarray-outlier-analysis.

Sporadic colorectal cancer (CRC) is a common malignancy and also one of the main causes of cancer deaths worldwide. Aberrant expression of the transcription factor SOX2 has recently been observed in several cancer types, but its role in CRC has not been fully elucidated. Here we studied the expression of SOX2 in 441 CRC patients by immunohistochemistry and related the expression to clinicopathological and molecular variables and patient prognosis. SOX2 was expressed in 11% of the tumors and was significantly associated to BRAFV600E mutation, but not to KRAS mutations (codon 12 and 13). SOX2 positivity was correlated to poor patient survival, especially in BRAFV600E mutated cases. In vitro studies showed that cells expressing the constitutively active BRAFV600E had increased SOX2 expression, a finding not found in cells expressing KRASG12V. Furthermore, blocking downstream BRAF signalling using a MEK-inhibitor resulted in a decreased expression of SOX2. Since SOX2 overexpression has been correlated to increased migration and invasion, we investigated the SOX2 expression in human CRC liver metastasis and found that a SOX2 positive primary CRC also had SOX2 expression in corresponding liver metastases. Finally we found that cells overexpressing SOX2 in vitro showed enhanced expression of FGFR1, which has been reported to correlate with liver metastasis in CRC. Our novel findings suggest that SOX2 expression is partly regulated by BRAF signalling, and an increased SOX2 expression may promote CRC metastasis and mediate a poor patient prognosis.

BackgroundGene variants known to contribute to Autoimmune Addison's disease (AAD) susceptibility include those at the MHC, MICA, CIITA, CTLA4, PTPN22, CYP27B1, NLRP-1 and CD274 loci. The majority of the genetic component to disease susceptibility has yet to be accounted for.AimTo investigate the role of 19 candidate genes in AAD susceptibility in six European case-control cohorts.MethodsA sequential association study design was employed with genotyping using Sequenom iPlex technology. In phase one, 85 SNPs in 19 genes were genotyped in UK and Norwegian AAD cohorts (691 AAD, 715 controls). In phase two, 21 SNPs in 11 genes were genotyped in German, Swedish, Italian and Polish cohorts (1264 AAD, 1221 controls). In phase three, to explore association of GATA3 polymorphisms with AAD and to determine if this association extended to other autoimmune conditions, 15 SNPs in GATA3 were studied in UK and Norwegian AAD cohorts, 1195 type 1 diabetes patients from Norway, 650 rheumatoid arthritis patients from New Zealand and in 283 UK Graves' disease patients. Meta-analysis was used to compare genotype frequencies between the participating centres, allowing for heterogeneity.ResultsWe report significant association with alleles of two STAT4 markers in AAD cohorts (rs4274624: P = 0.00016; rs10931481: P = 0.0007). In addition, nominal association of AAD with alleles at GATA3 was found in 3 patient cohorts and supported by meta-analysis. Association of AAD with CYP27B1 alleles was also confirmed, which replicates previous published data. Finally, nominal association was found at SNPs in both the NF-κB1 and IL23A genes in the UK and Italian cohorts respectively.ConclusionsVariants in the STAT4 gene, previously associated with other autoimmune conditions, confer susceptibility to AAD. Additionally, we report association of GATA3 variants with AAD: this adds to the recent report of association of GATA3 variants with rheumatoid arthritis.

AIMS/HYPOTHESIS: Diabetes mellitus is one of the most common endocrine disorders in dogs and is commonly proposed to be of autoimmune origin. Although the clinical presentation of human type 1 diabetes (T1D) and canine diabetes are similar, the aetiologies may differ. The aim of this study was to investigate if autoimmune aetiology resembling human T1D is as prevalent in dogs as previously reported. METHODS: Sera from 121 diabetic dogs representing 40 different breeds were tested for islet cell antibodies (ICA) and GAD65 autoantibodies (GADA) and compared with sera from 133 healthy dogs. ICA was detected by indirect immunofluorescence using both canine and human frozen sections. GADA was detected by in vitro transcription and translation (ITT) of human and canine GAD65, followed by immune precipitation. Sections of pancreata from five diabetic dogs and two control dogs were examined histopathologically including immunostaining for insulin, glucagon, somatostatin and pancreas polypeptide. RESULTS: None of the canine sera analysed tested positive for ICA on sections of frozen canine or human ICA pancreas. However, serum from one diabetic dog was weakly positive in the canine GADA assay and serum from one healthy dog was weakly positive in the human GADA assay. Histopathology showed marked degenerative changes in endocrine islets, including vacuolisation and variable loss of immune-staining for insulin. No sign of inflammation was noted. CONCLUSIONS/INTERPRETATIONS: Contrary to previous observations, based on results from tests for humoral autoreactivity towards islet proteins using four different assays, and histopathological examinations, we do not find any support for an islet autoimmune aetiology in canine diabetes mellitus.

We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.

Thymidine biosynthesis is essential in all cells. Inhibitors of the enzymes involved in this pathway (e.g. methotrexate) are thus frequently used as cytostatics. Due to its pivotal role in mycobacterial thymidylate synthesis dUTPase, which hydrolyzes dUTP into the dTTP precursor dUMP, has been suggested as a target for new antitubercular agents. All mycobacterial genomes encode dUTPase with a mycobacteria-specific surface loop absent in the human dUTPase. Using Mycobacterium smegmatis as a fast growing model for Mycobacterium tuberculosis, we demonstrate that dUTPase knock-out results in lethality that can be reverted by complementation with wild-type dUTPase. Interestingly, a mutant dUTPase gene lacking the genus-specific loop was unable to complement the knock-out phenotype. We also show that deletion of the mycobacteria-specific loop has no major effect on dUTPase enzymatic properties in vitro and thus a yet to be identified loop-specific function seems to be essential within the bacterial cell context. In addition, here we demonstrated that Mycobacterium tuberculosis dUTPase is fully functional in Mycobacterium smegmatis as it rescues the lethal knock-out phenotype. Our results indicate the potential of dUTPase as a target for antitubercular drugs and identify a genus-specific surface loop on the enzyme as a selective target.

Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only

A functional role of microRNAs (miRNAs or miRs) in neoplasia and metastasis is becoming clear, and the miR-200 family has received much attention for potentially regulating tumor progression. The miRNAs of this family have been shown to suppress epithelial-mesenchymal transition, and their down-regulation in some tumors promotes invasion and metastasis. Interestingly, while miR-200 is down-regulated in some cancers, it is up-regulated in others.We show that levels of miR-200 are increased in melanoma cell lines compared to normal melanocytes and that miR-200 family members play a role in determining modes of tumor cell migration. Individual tumor cells can invade in either elongated, "mesenchymal-type" or rounded, "amoeboid-like" modes and these two modes of invasion are inter-convertible [1]. In melanoma cell lines, expression of miR-200 members does not suppress invasion but rather leads to a switch between modes of invasion. MicroRNA-200c results in a higher proportion of cells adopting the rounded, amoeboid-like mode of invasion, while miR-200a results in a protrusion-associated elongated mode of invasion. Functional target identification studies suggest that the morphological effects of miR-200c may be mediated by reduced expression of MARCKS, which has been linked to formation of cell protrusions. In contrast miR-200a reduces actomyosin contractility, a feature of rounded morphology.Overall our findings call into question the general role of miR-200 in suppressing invasion and metastasis, and highlight novel distinguishing characteristics of individual miR-200 family members.

BACKGROUND: We have previously shown that vaccination with DNA encoding the encephalitogenic peptide myelin oligodendrocyte glycoprotein (MOG)(91-108) (pMOG) suppresses MOG(91-108)-induced rat Experimental Autoimmune Encephalomyelitis (EAE), a model for human Multiple Sclerosis (MS). The suppressive effect of pMOG is dependent on inclusion of CpG DNA in the plasmid backbone and is associated with early induction of Interferon (IFN)-beta. PRINCIPAL FINDINGS: In this study we examined the mechanisms underlying pMOG-induced protection. We found that in the DNA vaccinated cohort proinflammatory Interleukin (IL)-17 and IL-21 responses were dramatically reduced compared to in the control group, but that the expression of Foxp3 and Tumor Growth Factor (TGF)-beta1, which are associated with regulatory T cells, was not enhanced. Moreover, genes associated with Type I IFNs were upregulated. To delineate the role of IFN-beta in the protective mechanism we employed short interfering RNA (siRNA) to IFN-beta in the DNA vaccine. SiRNA to IFN-beta completely abrogated the protective effects of the vaccine, demonstrating that a local early elaboration of IFN-beta is important for EAE protection. IL-17 responses comparable to those in control rats developed in rats injected with the IFN-beta-silencing DNA vaccine. CONCLUSIONS: We herein demonstrate that DNA vaccination protects from proinflammatory Th17 cell responses during induction of EAE. The mechanism involves IFN-beta as IL-17 responses are rescued by silencing of IFN-beta during DNA vaccination.