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The endeavor to understand the human brain has seen more progress in the last few decades than in the previous two millennia. Still, our understanding of how the human brain relates to behavior in the real world and how this link is modulated by biological, social, and environmental factors is limited. To address this, we designed the Healthy Brain Study (HBS), an interdisciplinary, longitudinal, cohort study based on multidimensional, dynamic assessments in both the laboratory and the real world. Here, we describe the rationale and design of the currently ongoing HBS. The HBS is examining a population-based sample of 1,000 healthy participants (age 30–39) who are thoroughly studied across an entire year. Data are collected through cognitive, affective, behavioral, and physiological testing, neuroimaging, bio-sampling, questionnaires, ecological momentary assessment, and real-world assessments using wearable devices. These data will become an accessible resource for the scientific community enabling the next step in understanding the human brain and how it dynamically and individually operates in its bio-social context. An access procedure to the collected data and bio-samples is in place and published on https://www.healthybrainstudy.nl/en/data-and-methods/access. Trail registration: https://www.trialregister.nl/trial/7955.

Introduction The incidence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections in the Belgian community is mainly estimated based on test results of patients with coronavirus disease (COVID-19)-like symptoms. The aim of this study was to investigate the evolution of the SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) positivity ratio and distribution of viral loads within a cohort of asymptomatic patients screened prior hospitalization or surgery, stratified by age category. Materials/Methods We retrospectively studied data on SARS-CoV-2 real-time RT-PCR detection in respiratory tract samples of asymptomatic patients screened pre-hospitalization or pre-surgery in nine Belgian hospitals located in Flanders over a 12-month period (1 April 2020–31 March 2021). Results In total, 255925 SARS-CoV-2 RT-PCR test results and 2421 positive results for which a viral load was reported, were included in this study. An unweighted overall SARS-CoV-2 real-time RT-PCR positivity ratio of 1.27% was observed with strong spatiotemporal differences. SARS-CoV-2 circulated predominantly in 80+ year old individuals across all time periods except between the first and second COVID-19 wave and in 20–30 year old individuals before the second COVID-19 wave. In contrast to the first wave, a significantly higher positivity ratio was observed for the 20–40 age group in addition to the 80+ age group compared to the other age groups during the second wave. The median viral load follows a similar temporal evolution as the positivity rate with an increase ahead of the second wave and highest viral loads observed for 80+ year old individuals. Conclusion There was a high SARS-CoV-2 circulation among asymptomatic patients with a predominance and highest viral loads observed in the elderly. Moreover, ahead of the second COVID-19 wave an increase in median viral load was noted with the highest overall positivity ratio observed in 20–30 year old individuals, indicating they could have been the hidden drivers of this wave.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus., Author summary Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving disease pathology. In this study, we aimed to determine if genetic variation of ECE1 existed across a collection of C. albicans clinical isolates. Using a targeted sequencing approach, we identified a conserved variant candidalysin isoform. We then constructed isogenic chimeric strains harboring swapped Ece1p peptides or HiBiT tags between representative isoforms and showed impaired secretion with the variant that was associated with reduced virulence in vitro and in vivo. We also elucidated a conserved pattern between key sequences near lysine-arginine cleavage sites involved in Ece1p polypeptide processing by the Kex2p enzyme that correlate with predicted and experimental candidalysin secretion. Therefore, we present a novel mechanism for attenuation of C. albicans virulence at the ECE1 locus that may partly underlie differential clinical outcomes and contribute to the balance between commensal and pathogen at the vaginal mucosa.

Contains fulltext : 229469.pdf (Publisher’s version ) (Open Access) The aim of this study was to capture and understand the immediate recovery journey of patients following lumbar spinal fusion surgery and explore the interacting constructs that shape their journey. A qualitative study using Interpretive Phenomenological Analysis (IPA) approach. A purposive sample of 43 adult patients (≥16 years) undergoing ≤4 level instrumented fusion for back and/or leg pain of degenerative cause, were recruited pre-surgery from 4 UK spinal surgery centres. Patients completed a weekly diary expressed in their own words for the first 4 weeks following surgery to capture their life as lived. Diary content was based on previous research findings and recorded progress, recovery, motivation, symptoms, medications, healthcare appointments, rehabilitation, positive/negative thoughts, and significant moments; comparing to the previous week. To maximise completion and data quality, diaries could be completed in paper form, word document, as online survey or as audio recording. Strategies to enhance diary adherence included a weekly prompt. A framework analysis for individual diaries and then across participants (deductive and inductive components) captured emergent themes. Trustworthiness was enhanced by strategies including reflexivity, attention to negative cases and use of critical co-investigators. Twenty-eight participants (15 female; n = 18 (64.3%) aged 45-64) contributed weekly diaries (12 withdrew post-surgery, 3 did not follow through with surgery). Adherence with diaries was 89.8%. Participants provided diverse and vivid descriptions of recovery experiences. Three distinct recovery trajectories were identified: meaningful recovery (engagement in physical and functional activities to return to functionality/mobility); progressive recovery (small but meaningful improvement in physical ability with increasing confidence); and disruptive recovery (limited purpose for meaningful recovery). Important interacting constructs shaped participants' recovery including their pain experience and self-efficacy. This is the first account of immediate recovery trajectories from patients' perspectives. Recognition of a patient's trajectory may inform patient-centred recovery, follow-up and rehabilitation to improve patient outcomes.

Soil sealing increases surface runoff in a watershed and decreases infiltration into the soil. Consequently, urbanization poses a significant challenge for watershed management to mitigate faster runoff accumulation downstream and associated floods. Hydrological models are often employed to assess the impact of land-use dynamics on flood events. Alternatively, data-driven approaches combining time series of land use geodatasets and georeferenced flooded zones also allow to assess the relationship between soil sealing and flood severity. This study presents such data-driven analysis using a spatially explicit archive of flooded areas dating back to 1988 in the Flanders region of Belgium, which is characterized by urban sprawl. This archived data, along with time series of rainfall and land use, were analyzed for three middle-sized river subbasins using two machine learning methods: boosted regression trees and support vector regression. The machine learning methods were found suitable for this type of analysis, since their flexibility allows for spatially explicit models with larger sample sizes. However, the relationship between soil sealing and flood volume and extent could not be conclusively confirmed by our models. This may be due to data limitations, such as the limited number of recorded historical floods, inaccuracies in recorded historical flood polygons and inconsistencies in the land use classifications. It is therefore stressed that continued consistent monitoring of floods and land use changes is required. ispartof: PLOS ONE vol:15 issue:10 ispartof: location:United States status: published

In the aftermath of shock events, policy responses tend to be crafted under significant time constraints and high levels of uncertainty. The extent to which individuals comply with different policy designs can further influence how effective the policy responses are and how equitably their impacts are distributed in the population. Tools which allow policymakers to model different crisis trajectories, policy responses, and behavioral scenariosex antecan provide crucial timely support in the decision-making process. Set in the context of COVID-19 shelter in place policies, in this paper we present the COVID-19 Policy Evaluation (CoPE) tool, which is an agent-based modeling framework that enables researchers and policymakers to anticipate the relative impacts of policy decisions. Specifically, this framework illuminates the extent to which policy design features and behavioral responsiveness influence the efficacy and equity of policy responses to shock events. We show that while an early policy response can be highly effective, the impact of the timing is moderated by other aspects of policy design such as duration and targeting of the policy, as well as societal aspects such as trust and compliance among the population. More importantly, we show that even policies that are more effective overall can have disproportionate impacts on vulnerable populations. By disaggregating the impact of different policy design elements on different population groups, we provide an additional tool for policymakers to use in the design of targeted strategies for disproportionately affected populations.

Viruses have developed different strategies to escape from immune response. Among these, viral non-coding RNAs are invisible to the immune system and may affect the fate of the host cell. Bovine leukemia virus (BLV) encodes both short (miRNAs) and long (antisense AS1 and AS2) non-coding RNAs. To elucidate the mechanisms associated with BLV non-coding RNAs, we performed phenotypic and transcriptomic analyzes in a reverse genetics system. RNA sequencing of B-lymphocytes revealed that cell proliferation is the most significant mechanism associated with ablation of the viral non-coding RNAs. To assess the biological relevance of this observation, we determined the cell kinetic parameters in vivo using intravenous injection of BrdU and CFSE. Fitting the data to a mathematical model provided the rates of cell proliferation and death. Our data show that deletion of miRNAs correlates with reduced proliferation of the infected cell and lack of pathogenesis., Author summary BLV is a retrovirus that integrates into the genomic DNA of B-lymphocytes from a series of ruminant species (cattle, sheep, zebu, water buffalo and yack). Expression of viral proteins is almost undetectable in infected animals. In contrast, the BLV genome contains a cluster of 10 microRNAs that are abundantly transcribed in BLV-infected cells in vivo. In this report, we show that these microRNAs primarily regulate host cell proliferation. Ablation of the viral microRNAs affects BLV replication and suppresses leukemia development.

BackgroundPhrenic nerve injury (PNI) remains one of the most frequent complications during cryoballoon-based pulmonary vein isolation (CB-PVI). Since its introduction in 2013, the use of compound motor action potential (CMAP) for the prevention of PNI during CB-PVI is increasing; however, systematic outcome data are sparse.MethodsThe CMAP technique was applied in conjunction with abdominal palpation during pacing manoeuvres (10 mV, 2 ms) from the superior vena cava for 388 consecutive patients undergoing CB-PVI between January 2015 and May 2017 at our tertiary arrhythmia centre. Cryoablation was immediately terminated when CMAP amplitude was reduced by 30%.ResultsReductions in CMAP amplitude were observed in 16 (4%) of 388 patients during isolation of the right veins. Of these, 11 (69%) patients did not manifest a reduction in diaphragmatic excursions. The drop in CMAP amplitude was observed in 10 (63%) patients during ablation of the right superior pulmonary veins (PVs) and in 7 (44%) patients during ablation of the right inferior PVs. Postprocedural persistent PNI was observed in three of four patients for a duration of 6 months, with one of these patients remaining symptomatic at the 24-month follow-up. One of the four patients was lost to long-term follow-up.ConclusionsAll PNIs occurred during right-sided CB-PVI and were preceded by a reduction in CMAP amplitude. Thus, the standardized use of CMAP surveillance during CB-PVI is easily applicable, reliable and compared with other studies, results in a lower number of PNIs.

BackgroundKnowledge on the molecular epidemiology of Escherichia coli causing E. coli bacteremia (ECB) in the Netherlands is mostly based on extended-spectrum beta-lactamase-producing E. coli (ESBL-Ec). We determined differences in clonality and resistance and virulence gene (VG) content between non-ESBL-producing E. coli (non-ESBL-Ec) and ESBL-Ec blood isolates with different epidemiological characteristics.Materials/methodsA random selection of non-ESBL-Ec isolates as well as all available ESBL-Ec blood isolates was obtained from two Dutch hospitals between 2014 and 2016. Whole genome sequencing was performed to infer sequence types (STs), serotypes, acquired antibiotic resistance genes and VG scores, based on presence of 49 predefined putative pathogenic VG.ResultsST73 was most prevalent among the 212 non-ESBL-Ec (N=26, 12.3%) and ST131 among the 69 ESBL-Ec (N=30, 43.5%). Prevalence of ST131 among non-ESBL-Ec was 10.4% (N=22, P value < 0.001 compared to ESBL-Ec). O25:H4 was the most common serotype in both non-ESBL-Ec and ESBL-Ec. Median acquired resistance gene counts were 1 (IQR 1 – 6) and 7 (IQR 4 – 9) for non-ESBL-Ec and ESBL-Ec, respectively (P value < 0.001). Among non-ESBL-Ec, acquired resistance gene count was highest among blood isolates from a primary gastro-intestinal focus (median 4, IQR 1 – 8). Median VG scores were 13 (IQR 9 – 20) and 12 (IQR 8 – 14) for non-ESBL-Ec and ESBL-Ec, respectively (P value = 0.002). VG scores among non-ESBL-Ec from a primary urinary focus (median 15, IQR 11 – 21) were higher compared to non-ESBL-Ec from a primary gastro-intestinal (median 10, IQR 6 – 13) or hepatic-biliary focus (median 11, IQR 5 – 18) (P values = 0.007 and 0.036, respectively). VG content varied between different E. coli STs.ConclusionsNon-ESBL-Ec and ESBL-Ec blood isolates from two Dutch hospitals differed in clonal distribution, resistance gene and VG content. Also, resistance gene and VG content differed between non-ESBL-Ec from different primary foci of ECB.

IntroductionMeasuring the performance of a primary care system is one of the very first steps to find out whether there is room for improvement. To obtain an objective and comprehensive view, this measurement should come from both the supply and demand sides of the system. Patients' experiences of primary care have been studied around the world, but much less energy has been invested in researching providers' perspectives. This research aims to explore how primary care physicians working at commune health centers in Vietnam evaluate their performance and their opinions on how to improve the quality of primary care services.Materials and methodsFirst, a quantitative study was conducted using the validated Vietnamese PCAT questionnaire-provider expanded version (VN PCAT PE) targeting all primary care physicians (PCPs) working at commune health centers in a province of Central Vietnam. Next, a qualitative study was carried out, consisting of in-depth interviews with PCPs, to better understand the results of the quantitative survey and gain insight on barriers of primary care services and how to overcome them.ResultsIn the quantitative portion of our study, 150 PCPs rated the quality of ongoing care and first contact in CHCs as the best (3.09 and 3.11 out of 4, respectively), and coordination as the worst performing core domain (2.53). Twenty-two PCPs also participated in our qualitative research. In regards to challenges that primary care physicians face during their daily practice, three central themes emerged: 1) patient factors such as client attitude and knowledge, 2) provider factors such as the burden of administrative work and lack of training opportunities, and 3) contextual factors such as low income and lack of resources including medicines and diagnostics. Participants recommended more health promotion campaigns in the media, increasing the number of services available at CHCs (such as being able to take blood samples), reducing the workload related to administration for CHC leaders, greater government subsidies, and providing more training courses for PCPs.ConclusionsFindings from this study offer a valuable view from the supply-side of the primary care system, specifically those who directly deliver primary care services. Along with the earlier study on consumers' evaluation of the Vietnamese primary care system, and literature from other low and middle-income countries, these findings offer emerging evidence for policymakers to improve the quality of primary care in Vietnam.

Previous research shows conflicting findings for the effect of font readability on comprehension and memory for language. It has been found that—perhaps counterintuitively–a hard to read font can be beneficial for language comprehension, especially for difficult language. Here we test how font readability influences the subjective experience of poetry reading. In three experiments we tested the influence of poem difficulty and font readability on the subjective experience of poems. We specifically predicted that font readability would have opposite effects on the subjective experience of easy versus difficult poems. Participants read poems which could be more or less difficult in terms of conceptual or structural aspects, and which were presented in a font that was either easy or more difficult to read. Participants read existing poems and subsequently rated their subjective experience (measured through four dependent variables: overall liking, perceived flow of the poem, perceived topic clarity, and perceived structure). In line with previous literature we observed a Poem Difficulty x Font Readability interaction effect for subjective measures of poetry reading. We found that participants rated easy poems as nicer when presented in an easy to read font, as compared to when presented in a hard to read font. Despite the presence of the interaction effect, we did not observe the predicted opposite effect for more difficult poems. We conclude that font readability can influence reading of easy and more difficult poems differentially, with strongest effects for easy poems.

The acoustic properties of vocalizations in common marmosets differ between populations. These differences may be the result of social vocal learning, but they can also result from environmental or genetic differences between populations. We performed translocation experiments to separately quantify the influence of a change in the physical environment (experiment 1), and a change in the social environment (experiment 2) on the acoustic properties of calls from individual captive common marmosets. If population differences were due to genetic differences, we expected no change in the vocalizations of the translocated marmosets. If differences were due to environmental factors, we expected vocalizations to permanently change contingent with environmental changes. If social learning was involved, we expected that the vocalizations of animals translocated to a new population with a different dialect would become more similar to the new population. In experiment 1, we translocated marmosets to a different physical environment without changing the social composition of the groups or their neighbours. Immediately after the translocation to the new facility, one out of three call types showed a significant change in call structure, but 5-6 weeks later, the calls were no longer different from before the translocation. Thus, the novel physical environment did not induce long lasting changes in the vocalizations of the marmosets. In experiment 2, we translocated marmosets to a new population with a different dialect. Importantly, our previous work had shown that these two populations differed significantly in vocalization structure. The translocated marmosets were still housed in their original social group, but after translocation they were surrounded by the vocalizations from neighbouring groups of the new population. The vocal distance between the translocated individuals and the new population decreased for two out of three call types over 16 weeks. Thus, even without direct social contact or interaction, the vocalizations of the translocated animals converged towards the new population, indicating that common marmosets can modify their calls due to acoustic input from conspecifics alone, via crowd vocal learning. To our knowledge, this is the first study able to distinguish between different explanations for vocal dialects as well as to show crowd vocal learning in a primate species.

In Mycobacterium tuberculosis (Mtb) the detection of single nucleotide polymorphisms (SNPs) is of high importance both for diagnostics, since drug resistance is primarily caused by the acquisition of SNPs in multiple drug targets, and for epidemiological studies in which strain typing is performed by SNP identification. To provide the necessary coverage of clinically relevant resistance profiles and strain types, nucleic acid-based measurement techniques must be able to detect a large number of potential SNPs. Since the Mtb problem is pressing in many resource-poor countries, requiring low-cost point-of-care biosensors, this is a non-trivial technological challenge. This paper presents a proof-of-concept in which we chose simple DNA-DNA hybridization as a sensing principle since this can be transferred to existing low-cost hardware platforms, and we pushed the multiplex boundaries of it. With a custom designed probe set and a physicochemical-driven data analysis it was possible to simultaneously detect the presence of SNPs associated with first- and second-line drug resistance and Mtb strain typing. We have demonstrated its use for the identification of drug resistance and strain type from a panel of phylogenetically diverse clinical strains. Furthermore, reliable detection of the presence of a minority population (

BackgroundIn non-small cell lung cancer (NSCLC), PD-L1 expression on either tumor cells (TC) or both TC and tumor-infiltrating immune cells (IC) is currently the most used biomarker in cancer immunotherapy. However, the mechanisms involved in PD-L1 regulation are not fully understood. To provide better insight in these mechanisms, a multiangular analysis approach was used to combine protein and mRNA expression with several clinicopathological characteristics.Patients and methodsArchival tissues from 640 early stage, resected NSCLC patients were analyzed with immunohistochemistry for expression of PD-L1 and CD8 infiltration. In addition, mutational status and expression of a selection of immune genes involved in the PD-L1/PD-1 axis and T-cell response was determined.ResultsTumors with high PD-L1 expression on TC or on IC represent two subsets of NSCLC with minimal overlap. We observed that PD-L1 expression on IC irrespective of expression on TC is a good marker for inflammation within tumors. In the tumors with the highest IC expression and absent TC expression an association with reduced IFNγ downstream signaling in tumor cells was observed.ConclusionsThese results show that PD-L1 expression on TC and IC are both independent hallmarks of the inflamed phenotype in NSCLC, and TC-negative/IC-high tumors can also be categorized as inflamed. The lack of correlation between PD-L1 TC and IC expression in this subgroup may be caused by impaired IFNγ signaling in tumor cells. These findings may bring a better understanding of the tumor-immune system interaction and the clinical relevance of PD-L1 expression on IC irrespective of PD-L1 expression on TC.

INTRODUCTION: It is crucial to understand the factors that introduce variability before applying metabolomics to clinical and biomarker research.OBJECTIVES: We quantified technical and biological variability of both fasting and postprandial metabolite concentrations measured using 1H NMR spectroscopy in plasma samples.METHODS: In the Netherlands Epidemiology of Obesity study (n = 6,671), 148 metabolite concentrations (101 metabolites belonging to lipoprotein subclasses) were measured under fasting and postprandial states (150 minutes after a mixed liquid meal). Technical variability was evaluated among 265 fasting and 851 postprandial samples, with the identical blood plasma sample being measured twice by the same laboratory protocol. Biological reproducibility was assessed by measuring 165 individuals twice across time for evaluation of short- (3 years) biological variability. Intra-class coefficients (ICCs) were used to assess variability. The ICCs of the fasting metabolites were compared with the postprandial metabolites using two-sided paired Wilcoxon test separately for short- and long-term measurements.RESULTS: Both fasting and postprandial metabolite concentrations showed high technical reproducibility using 1H NMR spectroscopy (median ICC = 0.99). Postprandial metabolite concentrations revealed slightly higher ICC scores than fasting ones in short-term repeat measures (median ICC in postprandial and fasting metabolite concentrations 0.72 versus 0.67, Wilcoxon p-value = 8.0×10-14). Variability did not increase further in a long-term repeat measure, with median ICC in postprandial of 0.64 and in fasting metabolite concentrations 0.66.CONCLUSION: Technical reproducibility is excellent. Biological reproducibility of postprandial metabolite concentrations showed a less or equal variability than fasting metabolite concentrations over time.

Combining different metabolomics platforms can contribute significantly to the discovery of complementary processes expressed under different conditions. However, analysing the fused data might be hampered by the difference in their quality. In metabolomics data, one often observes that measurement errors increase with increasing measurement level and that different platforms have different measurement error variance. In this paper we compare three different approaches to correct for the measurement error heterogeneity, by transformation of the raw data, by weighted filtering before modelling and by a modelling approach using a weighted sum of residuals. For an illustration of these different approaches we analyse data from healthy obese and diabetic obese individuals, obtained from two metabolomics platforms. Concluding, the filtering and modelling approaches that both estimate a model of the measurement error did not outperform the data transformation approaches for this application. This is probably due to the limited difference in measurement error and the fact that estimation of measurement error models is unstable due to the small number of repeats available. A transformation of the data improves the classification of the two groups. ispartof: PLOS ONE vol:13 issue:4 ispartof: location:United States status: published

Swine dysentery (SD) is an economically important diarrheal disease in pigs caused by different strongly hemolytic Brachyspira (B.) species, such as B. hyodysenteriae, B. suanatina and B. hampsonii. Possible associations of epidemiologic data, such as multilocus sequence types (STs) to virulence gene profiles and antimicrobial susceptibility are rather scarce, particularly for B. hyodysenteriae isolates from Germany. In this study, B. hyodysenteriae (n = 116) isolated from diarrheic pigs between 1990 and 2016 in Germany were investigated for their STs, susceptibility to the major drugs used for treatment of SD (tiamulin and valnemulin) and genes that were previously linked with virulence and encode for hemolysins (tlyA, tlyB, tlyC, hlyA, BHWA1_RS02885, BHWA1_RS09085, BHWA1_RS04705, and BHWA1_RS02195), outer membrane proteins (OMPs) (bhlp16, bhlp17.6, bhlp29.7, bhmp39f, and bhmp39h) as well as iron acquisition factors (ftnA and bitC). Multilocus sequence typing (MLST) revealed that 79.4% of the isolates belonged to only three STs, namely ST52 (41.4%), ST8 (12.1%), and ST112 (25.9%) which have been observed in other European countries before. Another 24 isolates belonged to twelve new STs (ST113-118, ST120-123, ST131, and ST193). The temporal distribution of STs revealed the presence of new STs as well as the regular presence of ST52 over three decades (1990s–2000s). The proportion of strains that showed resistance to both tiamulin und valnemulin (39.1%) varied considerably among the most frequent STs ranging from 0% (0/14 isolates resistant) in ST8 isolates to 46.7% (14/30), 52.1% (25/48), and 85.7% (6/7) in isolates belonging to ST112, ST52, and ST114, respectively. All hemolysin genes as well as the iron-related gene ftnA and the OMP gene bhlp29.7 were regularly present in the isolates, while the OMP genes bhlp17.6 and bhmp39h could not be detected. Sequence analysis of hemolysin genes of selected isolates revealed co-evolution of tlyB, BHWA1_RS02885, BHWA1_RS09085, and BHWA1_RS02195 with the core genome and suggested independent evolution of tlyA, tlyC, and hlyA. Our data indicate that in Germany, swine dysentery might be caused by a limited number of B. hyodysenteriae clonal groups. Major STs (ST8, ST52, and ST112) are shared with other countries in Europe suggesting a possible role of the European intra-Community trade of pigs in the dissemination of certain clones. The identification of several novel STs, some of which are single or double locus variants of ST52, may on the other hand hint towards an ongoing diversification of the pathogen in the studied area. The linkage of pleuromutilin susceptibility and sequence type of an isolate might reflect a clonal expansion of the underlying resistance mechanism, namely mutations in the ribosomal RNA genes. A linkage between single virulence-associated genes (VAGs) or even VAG patterns and the phylogenetic background of the isolates could not be established, since almost all VAGs were regularly present in the isolates.

ObjectiveQuantification software for coronary calcification is often used to measure abdominal aortic calcification on computed tomography (CT) images. However, there is no evidence substantiating the reliability and accuracy of these tools in this setting. Differences in coronary and abdominal CT acquisition and presence of intravascular contrast may affect the results of these tools. Therefore, this study investigates the effects of CT acquisition parameters and iodine contrast on automated quantification of aortic calcium on CT.MethodsCalcium scores, provided in volume and mass, were assessed by automated calcium quantification software on CT scans. First, differences in calcium scores between the abdominal and coronary CT scanning protocols were assessed by imaging a thorax phantom containing calcifications of 9 metrical variations. Second, aortic calcification was quantified in 50 unenhanced and contrast-enhanced clinical abdominal CT scans at a calcification threshold of 299 Hounsfield Units (HU). Also, the lowest possible HU threshold for calcifications was calculated per individual patient and compared to a 130 HU threshold between contrast enhanced and unenhanced CT images, respectively.ResultsNo significant differences in volume and mass scores between the abdominal and the coronary CT protocol were found. However, volume and mass of all calcifications were overestimated compared to the physical volume and mass (volume range: 0-649%; mass range: 0-2619%). In comparing unenhanced versus contrast-enhanced CT images showed significant volume differences for both thresholds, as well as for mass differences for the 130 vs patient-specific threshold (230 +/- 22.6 HU).ConclusionCalcification scoring on CT angiography tends to grossly overestimate volume and mass suggesting a low accuracy and reliability. These are reduced further by interference of intravascular contrast. Future studies applying calcium quantification tools on CT angiography imaging should acknowledge these issues and apply corrective measures to ensure the validity of their outcomes.

Background Body fat distribution is, next to overall obesity, an important risk factor for cardiometabolic outcomes in the general population. In particular, visceral adipose tissue (VAT) is strongly associated with cardiometabolic risk factors. Since it is unclear whether body fat distribution is also important in men and women with obesity we investigated the associations between measures of body fat distribution and cardiometabolic risk factors in men and women with obesity. Methods In this cross-sectional analysis of obese men and women (BMI≥30 kg/m2) included in the Netherlands Epidemiology of Obesity Study, waist:hip ratio(WHR), waist circumference, and MRI-based abdominal subcutaneous adipose tissue (aSAT) and VAT were determined. Associations between measures of body fat distribution and presence of ≥1 risk factor, such as hypertension or hypertriglyceridemia, were examined using logistic regression analyses; stratified by sex and adjusted for age, ethnicity, education, tobacco smoking, alcohol consumption, physical activity and depending on the association additionally for total body fat or VAT. Results We included 2,983 obese individuals (57% women) with a mean age of 56 and standard deviation (SD) of 6 and mean BMI of 34.0 kg/m2 (4.0), after exclusion of individuals with missing values of cardiometabolic risk factors (n = 33). 241 individuals were obese without other cardiometabolic risk factors. In obese women, all measures of body fat distribution except aSAT (OR per SD:0.76, 95%CI: 0.53, 1.10) were associated with having ≥1 cardiometabolic risk factor, of which VAT most strongly associated (5.77; 3.02, 11.01). In obese men, associations of body fat distribution and the presence of cardiometabolic risk factors were attenuated. (e.g. VAT:1.42; 0.84, 2.41). Conclusions In obese women, but less so in men, measures of body fat distribution, of which VAT most strongly, are associated with cardiometabolic risk factors.

Contains fulltext : 173550.pdf (Publisher’s version ) (Open Access) Language comprehension involves the simultaneous processing of information at the phonological, syntactic, and lexical level. We track these three distinct streams of information in the brain by using stochastic measures derived from computational language models to detect neural correlates of phoneme, part-of-speech, and word processing in an fMRI experiment. Probabilistic language models have proven to be useful tools for studying how language is processed as a sequence of symbols unfolding in time. Conditional probabilities between sequences of words are at the basis of probabilistic measures such as surprisal and perplexity which have been successfully used as predictors of several behavioural and neural correlates of sentence processing. Here we computed perplexity from sequences of words and their parts of speech, and their phonemic transcriptions. Brain activity time-locked to each word is regressed on the three model-derived measures. We observe that the brain keeps track of the statistical structure of lexical, syntactic and phonological information in distinct areas. 18 p.

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.

It has been demonstrated that hepatic apparent diffusion coefficients (ADC) are decreasing in patients with a Fontan circulation. It remains however unclear whether this is a true decrease of molecular diffusion, or rather reflects decreased microperfusion due to decreased portal blood flow. The purpose of this study was therefore to differentiate diffusion and microperfusion using intravoxel incoherent motion (IVIM) modeled diffusion-weighted imaging (DWI) for different liver segments in patients with a Fontan circulation, compare to a control group, and relate with liver function, chronic hepatic congestion and hepatic disease. For that purpose, livers of 59 consecutively included patients with Fontan circulation (29 men; mean-age, 19.1 years) were examined (Oct 2012-Dec 2013) with 1.5T MRI and DWI (b = 0,50,100,250,500, 750,1500,1750 s/mm(2)). IVIM (D-slow,D-fast,f(fast)) and ADC were calculated for eight liver segments, compared to a control group (19 volunteers; 10 men; mean-age, 32.9 years), and correlated to follow-up duration, clinical variables, and laboratory measurements associated with liver function. The results demonstrated that microperfusion was reduced (p

Contains fulltext : 174709.pdf (Publisher’s version ) (Open Access) Experimental bone marrow transplantation (BMT) in mice is commonly used to assess the role of immune cell-specific genes in various pathophysiological settings. The application of BMT in obesity research is hampered by the significant reduction in high-fat diet (HFD)-induced obesity. We set out to characterize metabolic tissues that may be affected by the BMT procedure and impair the HFD-induced response. Male C57BL/6 mice underwent syngeneic BMT using lethal irradiation. After a recovery period of 8 weeks they were fed a low-fat diet (LFD) or HFD for 16 weeks. HFD-induced obesity was reduced in mice after BMT as compared to HFD-fed control mice, characterized by both a reduced fat (-33%; p

Background\ud \ud Chorioamnionitis can induce pulmonary inflammation and promote bronchopulmonary dysplasia development, distinguished by alveolar simplification and impaired vascular growth. Chorioamnionitis is more common during the extremely preterm canalicular lung stage (crucial for vascular development); and increases the risk for subsequent sepsis. We hypothesized that single/combined exposure to chronic and/or acute inflammation induces pulmonary inflammatory responses and vascular changes.\ud \ud Methods\ud \ud Ovine fetuses were intra-amniotically exposed to chronic Ureaplasma parvum (UP) at 24 days (d) before extreme preterm delivery at 94d (term 147d) and/or to lipopolysaccharide (LPS) 7 or 2d before delivery. Pulmonary inflammation, vascular remodeling and angiogenic factors were assessed.\ud \ud Results\ud \ud LPS exposure increased CD3-positive and myeloperoxidase-positive cells. Combined UP-LPS exposure increased pulmonary inflammation compared with 2d LPS or UP groups. The UP+2d LPS group had an increased adventitial fibrosis score when compared with UP-treated animals. A reduced wall-to-lumen ratio was found in the 7d LPS animals when compared to the 2d LPS-treated animals. Exposure to UP+2d LPS reduced VEGF and VEGFR-2 levels compared with 2d LPS-treated animals. Angiopoietin-1 (Ang1) and tunica interna endothelial cell kinase 2 (Tie-2) levels were decreased after UP+7d LPS as well as after 7d LPS, but not with UP alone.\ud \ud Conclusion\ud \ud Chronic UP and subsequent LPS exposure increased pulmonary inflammation and decreased expression of angiogenic growth factors and receptors when compared to single hit-exposed animals.\ud \ud Figures

Many daily life activities demand precise integration of spatial and temporal information of sensory inputs followed by appropriate motor actions. This type of integration is carried out in part by the cerebellum, which has been postulated to play a central role in learning and timing of movements. Cerebellar damage due to atrophy or lesions may compromise forward-model processing, in which both spatial and temporal cues are used to achieve prediction for future motor states. In the present study we sought to further investigate the cerebellar contribution to predictive and reactive motor timing, as well as to learning of sequential order and temporal intervals in these tasks. We tested patients with spinocerebellar ataxia type 6 (SCA6) and healthy controls for two related motor tasks; one requiring spatio-temporal prediction of dynamic visual stimuli and another one requiring reactive timing only. We found that healthy controls established spatio-temporal prediction in their responses with high temporal precision, which was absent in the cerebellar patients. SCA6 patients showed lower predictive motor timing, coinciding with a reduced number of correct responses during the ‘anticipatory’ period on the task. Moreover, on the task utilizing reactive motor timing functions, control participants showed both sequence order and temporal interval learning, whereas patients only showed sequence order learning. These results suggest that SCA6 affects predictive motor timing and temporal interval learning. Our results support and highlight cerebellar contribution to timing and argue for cerebellar engagement during spatio-temporal prediction of upcoming events.

Background Electrogram-based identification of the regions maintaining persistent Atrial Fibrillation (AF) is a subject of ongoing debate. Here, we explore the concept of local electrical dyssynchrony to identify AF drivers. Methods and Results Local electrical dyssynchrony was calculated using mean phase coherence. High-density epicardial mapping along with mathematical model were used to explore the link between local dyssynchrony and properties of wave conduction. High-density mapping showed a positive correlation between the dyssynchrony and number of fibrillatory waves (R2 = 0.68, p

This study investigates adaptation of ST406, a prevalent P. aeruginosa clone, present in 15% of chronically infected cystic fibrosis (CF) patients in the Netherlands, in a newly infected CF patient during three years using whole genome sequencing (WGS), transcriptomics, and phenotypic assays, including biofilm formation. WGS-based phylogeny demonstrates that ST406 is genetically distinct from other reported CF related strains or epidemic clones. Comparative genomic analysis of the early (S1) and late (S2) isolate yielded 42 single nucleotide polymorphisms (SNPs) and 10 indels and a single 7 kb genomic fragment only found in S2. Most SNPs and differentially expressed genes encoded proteins involved in metabolism, secretion and signal transduction or transcription. SNPs were identified in regulator genes mexT and exsA and coincided with differential gene expression of mexE and mexF, encoding the MexE/F efflux pump, genes encoding the type six secretion system (T6SS) and type three secretion system (T3SS), which have also been previously implicated in adaptation of other P. aeruginosa strains during chronic infection of CF lungs. The observation that genetically different strains from different patients have accumulated similar genetic adaptations supports the concept of adaptive parallel evolution of P. aeruginosa in chronically infected CF patients. Phenotypically, there was loss of biofilm maturation coinciding with a significant lower level of transcription of both bfmR and bfmS during chronic colonization. These data suggest that the high-prevalent Dutch CF clone ST406 displays adaptation to the CF lung niche, which involves a limited number of mutations affecting regulators controlling biofilm formation and secretion and genes involved in metabolism. These genes could provide good targets for anti-pseudomonal therapy.

BackgroundThe reduced ability to identify a second target when it is presented in close temporal succession of a first target is called the attentional blink (AB). Studies have shown large individual differences in AB task performance, where lower task performance has been associated with more reversed order reports of both targets if these were presented in direct succession. In order to study the suggestion that reversed order reports reflect loss of temporal information, in the current study, we investigated whether individuals with a larger AB have a higher tendency to temporally integrate both targets into one visual event by using an AB paradigm containing symbol target stimuli.Methodology/Principal FindingsIndeed, we found a positive relation between the tendency to temporally integrate information and individual AB magnitude. In contrast to earlier work, we found no relation between order reversals and individual AB magnitude. The occurrence of temporal integration was negatively related to the number of order reversals, indicating that individuals either integrated or separated and reversed information.ConclusionWe conclude that individuals with better AB task performance use a shorter time window to integrate information, and therefore have higher preservation of temporal information. Furthermore, order reversals observed in paradigms with alphanumeric targets indeed seem to at least partially reflect temporal integration of both targets. Given the negative relation between temporal integration and 'true' order reversals observed with the current symbolic target set, these two behavioral outcomes seem to be two sides of the same coin.

The Atlantic salmon (Salmo salar) and African bichir (Polypterus senegalus) are both actinopterygian fish species that continuously replace their teeth without the involvement of a successional dental lamina. Instead, they share the presence of a middle dental epithelium: an epithelial tier enclosed by inner and outer dental epithelium. It has been hypothesized that this tier could functionally substitute for a successional dental lamina and might be a potential niche to house epithelial stem cells involved in tooth cycling. Therefore, in this study we performed a BrdU pulse chase experiment on both species to (1) determine the localization and extent of proliferating cells in the dental epithelial layers, (2) describe cell dynamics and (3) investigate if label-retaining cells are present, suggestive for the putative presence of stem cells. Cells proliferate in the middle dental epithelium, outer dental epithelium and cervical loop at the lingual side of the dental organ to form a new tooth germ. Using long chase times, both in S. salar (eight weeks) and P. senegalus (eight weeks and twelve weeks), we could not reveal the presence of label-retaining cells in the dental organ. Immunostaining of P. senegalus dental organs for the transcription factor Sox2, often used as a stem cell marker, labelled cells in the zone of outer dental epithelium which grades into the oral epithelium (ODE transition zone) and the inner dental epithelium of a successor only. The location of Sox2 distribution does not provide evidence for epithelial stem cells in the dental organ and, more specifically, in the middle dental epithelium. Comparison of S. salar and P. senegalus reveals shared traits in tooth cycling and thus advances our understanding of the developmental mechanism that ensures lifelong replacement.

textabstractBackground Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. Method We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. Results Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5%concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. Conclusion Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

INTRODUCTION: Segregation of patients with cystic fibrosis (CF) was implemented to prevent chronic infection with epidemic Pseudomonas aeruginosa strains with presumed detrimental clinical effects, but its effectiveness has not been carefully evaluated. METHODS: The effect of strict segregation on the incidence of P. aeruginosa infection in CF patients was investigated through longitudinal protocolized follow-up of respiratory tract infection before and after segregation. In two nested cross-sectional studies in 2007 and 2011 the P. aeruginosa population structure was investigated and clinical parameters were determined in patients with and without infection with the Dutch epidemic P. aeruginosa clone (ST406). RESULTS: Of 784 included patients 315 and 382 were at risk for acquiring chronic P. aeruginosa infection before and after segregation. Acquisition rates were, respectively, 0.14 and 0.05 per 1,000 days at risk (HR: 0.66, 95% CI [0.2548-1.541]; p = 0.28). An exploratory subgroup analysis indicated lower acquisition after segregation in children < 15 years of age (HR: 0.43, 95% CI[0.21-0.95]; p = 0.04). P. aeruginosa population structure did not change after segregation and ST406 was not associated with lung function decline, death or lung transplantation. CONCLUSIONS: Strict segregation was not associated with a statistically significant lower acquisition of chronic P. aeruginosa infection and ST406 was not associated with adverse clinical outcome. After segregation there were no new acquisitions of ST406. In an unplanned exploratory analysis chronic acquisition of P. aeruginosa was lower after implementation of segregation in patients under 15 years of age.

Retroviruses are not expected to encode miRNAs because of the potential problem of self-cleavage of their genomic RNAs. This assumption has recently been challenged by experiments showing that bovine leukemia virus (BLV) encodes miRNAs from intragenomic Pol III promoters. The BLV miRNAs are abundantly expressed in B-cell tumors in the absence of significant levels of genomic and subgenomic viral RNAs. Using deep RNA sequencing and functional reporter assays, we show that miRNAs mediate the expression of genes involved in cell signaling, cancer and immunity. We further demonstrate that BLV miRNAs are essential to induce B-cell tumors in an experimental model and to promote efficient viral replication in the natural host., Author Summary The recent discovery of miRNA expression by retroviruses is a matter of active debate and some controversy. Several retroviruses including human immunodeficiency virus (HIV) potentially encode functional small non-coding RNAs that are nevertheless undetectable in vivo. While these miRNAs regulate multiple host and viral processes in vitro, their contribution to infection and pathogenesis remains largely unknown. Using next-generation sequencing and reverse genetics, we provide mechanistic and functional evidence demonstrating that BLV miRNAs are functional genetic elements that regulate replication and contribute to oncogenesis in suitable animal models.

Contains fulltext : 157640.pdf (Publisher’s version ) (Open Access) Personal pronouns have been shown to influence cognitive perspective taking during comprehension. Studies using single sentences found that 3rd person pronouns facilitate the construction of a mental model from an observer's perspective, whereas 2nd person pronouns support an actor's perspective. The direction of the effect for 1st person pronouns seems to depend on the situational context. In the present study, we investigated how personal pronouns influence discourse comprehension when people read fiction stories and if this has consequences for affective components like emotion during reading or appreciation of the story. We wanted to find out if personal pronouns affect immersion and arousal, as well as appreciation of fiction. In a natural reading paradigm, we measured electrodermal activity and story immersion, while participants read literary stories with 1st and 3rd person pronouns referring to the protagonist. In addition, participants rated and ranked the stories for appreciation. Our results show that stories with 1st person pronouns lead to higher immersion. Two factors - transportation into the story world and mental imagery during reading - in particular showed higher scores for 1st person as compared to 3rd person pronoun stories. In contrast, arousal as measured by electrodermal activity seemed tentatively higher for 3rd person pronoun stories. The two measures of appreciation were not affected by the pronoun manipulation. Our findings underscore the importance of perspective for language processing, and additionally show which aspects of the narrative experience are influenced by a change in perspective. 18 p.

Different animal models have been used to study the effects of prenatal protein undernutrition and the mechanisms by which these occur. In mammals, the maternal diet is manipulated, exerting both direct nutritional and indirect hormonal effects. Chicken embryos develop independent from the hen in the egg. Therefore, in the chicken, the direct effects of protein deficiency by albumen removal early during incubation can be examined. Prenatal protein undernutrition was established in layer-type eggs by the partial replacement of albumen by saline at embryonic day 1 (albumen-deprived group), compared to a mock-treated sham and a non-treated control group. At hatch, survival of the albumen-deprived group was lower compared to the control and sham group due to increased early mortality by the manipulation. No treatment differences in yolk-free body weight or yolk weight could be detected. The water content of the yolk was reduced, whereas the water content of the carcass was increased in the albumen-deprived group, compared to the control group, indicating less uptake of nutrients from the yolk. At embryonic day 16, 20 and at hatch, plasma triiodothyronine (T3), corticosterone, lactate or glucose concentrations and hepatic glycogen content were not affected by treatment. At embryonic day 20, the plasma thyroxine (T4) concentrations of the albumen-deprived embryos was reduced compared to the control group, indicating a decreased metabolic rate. Screening for differential protein expression in the liver at hatch using two-dimensional difference gel electrophoresis revealed not only changed abundance of proteins important for amino acid metabolism, but also of enzymes related to energy and glucose metabolism. Interestingly, GLUT1, a glucose transporter, and PCK2 and FBP1, two out of three regulatory enzymes of the gluconeogenesis were dysregulated. No parallel differences in gene expressions causing the differences in protein abundance could be detected pointing to post-transcriptional or post-translational regulation of the observed differences. ispartof: PLoS One vol:9 issue:4 pages:1-12 ispartof: location:United States status: published

Contains fulltext : 219525.pdf (Publisher’s version ) (Open Access) Objective: The Functional Assessment of Chronic Illness Therapy- Fatigue (FACIT-F) is commonly used to assess fatigue in rheumatic diseases, and has shown to discriminate better across levels of the fatigue spectrum than other commonly used measures. The aim of this study was to assess the cross-language measurement equivalence of the English, French, and Dutch versions of the FACIT-F in systemic sclerosis (SSc) patients. Methods: The FACIT-F was completed by 871 English-speaking Canadian, 238 French-speaking Canadian and 230 Dutch SSc patients. Confirmatory factor analysis was used to assess the factor structure in the three samples. The Multiple-Indicator Multiple-Cause (MIMIC) model was utilized to assess differential item functioning (DIF), comparing English versus French and versus Dutch patient responses separately. Results: A unidimensional factor model showed good fit in all samples. Comparing French versus English patients, statistically significant, but small-magnitude DIF was found for 3 of 13 items. French patients had 0.04 of a standard deviation (SD) lower latent fatigue scores than English patients and there was an increase of only 0.03 SD after accounting for DIF. For the Dutch versus English comparison, 4 items showed small, but statistically significant, DIF. Dutch patients had 0.20 SD lower latent fatigue scores than English patients. After correcting for DIF, there was a reduction of 0.16 SD in this difference. Conclusions: There was statistically significant DIF in several items, but the overall effect on fatigue scores was minimal. English, French and Dutch versions of the FACIT-F can be reasonably treated as having equivalent scoring metrics. 7 p.

Contains fulltext : 136017.pdf (Publisher’s version ) (Open Access) Opening of the mitochondrial permeability transition pore (mPTP) is involved in various cellular processes including apoptosis induction. Two distinct states of mPTP opening have been identified allowing the transfer of molecules with a molecular weight

Mitochondrial morphology and function are coupled in healthy cells, during pathological conditions and (adaptation to) endogenous and exogenous stress. In this sense mitochondrial shape can range from small globular compartments to complex filamentous networks, even within the same cell. Understanding how mitochondrial morphological changes (i.e. “mitochondrial dynamics”) are linked to cellular (patho) physiology is currently the subject of intense study and requires detailed quantitative information. During the last decade, various computational approaches have been developed for automated 2-dimensional (2D) analysis of mitochondrial morphology and number in microscopy images. Although these strategies are well suited for analysis of adhering cells with a flat morphology they are not applicable for thicker cells, which require a three-dimensional (3D) image acquisition and analysis procedure. Here we developed and validated an automated image analysis algorithm allowing simultaneous 3D quantification of mitochondrial morphology and network properties in human endothelial cells (HUVECs). Cells expressing a mitochondria-targeted green fluorescence protein (mitoGFP) were visualized by 3D confocal microscopy and mitochondrial morphology was quantified using both the established 2D method and the new 3D strategy. We demonstrate that both analyses can be used to characterize and discriminate between various mitochondrial morphologies and network properties. However, the results from 2D and 3D analysis were not equivalent when filamentous mitochondria in normal HUVECs were compared with circular/spherical mitochondria in metabolically stressed HUVECs treated with rotenone (ROT). 2D quantification suggested that metabolic stress induced mitochondrial fragmentation and loss of biomass. In contrast, 3D analysis revealed that the mitochondrial network structure was dissolved without affecting the amount and size of the organelles. Thus, our results demonstrate that 3D imaging and quantification are crucial for proper understanding of mitochondrial shape and topology in non-flat cells. In summary, we here present an integrative method for unbiased 3D quantification of mitochondrial shape and network properties in mammalian cells.

ObjectiveIn endovascular aortic aneurysm repair (EVAR), proximal type 1A endoleaks can occur as a result of hostile neck anatomy or over- or undersizing of the endograft. As the current standard is based on the diameter or average of the short and long axes in a central lumen reconstruction image, it can falter in irregularly shaped aortic necks. An alternative method is circumference-based, therefore minimizing the measurement error. In this study we aimed to assess the degree of discrepancy between both methods and the association of this discrepancy with the occurrence of endoleak type 1A.MethodsAll patients with early (ResultsIn 482 EVAR patients, 18 early endoleak type 1A cases were found (3.9%). After exclusion, 12 cases remained and 48 matching controls were found. No significant differences were found between the two measuring methods at any level below the renal arteries. The interobserver variability was significant for the D(mean) (0.4 +/- 1.69 mm, P = .02) and was larger than the D(circ) method (-0.1 +/- 1.03 mm, P = .35). In only four out of 12 cases the endograft size was 10-20% larger than the D(mean) and D(circ) measurements. The differences between the diameter of the D(mean) and D(circ) and the chosen endograft were smaller for the case group (-8 +/- 25.6% and -7 +/- 24%) than for the control group. (-12.4 +/- 12.4% and -11 +/- 10.7%).ConclusionThe difference between the D(mean) and D(circ) methods for aortic neck measurement was not large enough to play a significant role in the incidence of endoleak type 1A. Inadequate oversizing and considerable beta-angulation of the aortic neck may have been the cause of endoleak type 1A in this population. Robust and well-investigated sizing methods are paramount for accurate endograft sizing and prevention of endoleak type 1A. Therefore the lack of studies in this field and a sizeable inter-observer variability do not justify the widespread reliance on the traditional diameter-based methods for endograft sizing.

Contains fulltext : 138935.pdf (Publisher’s version ) (Open Access) Very low calorie diets (VLCD) with and without exercise programs lead to major metabolic improvements in obese type 2 diabetes patients. The mechanisms underlying these improvements have so far not been elucidated fully. To further investigate the mechanisms of a VLCD with or without exercise and to uncover possible biomarkers associated with these interventions, blood samples were collected from 27 obese type 2 diabetes patients before and after a 16-week VLCD (Modifast approximately 450 kcal/day). Thirteen of these patients followed an exercise program in addition to the VCLD. Plasma was obtained from 27 lean and 27 obese controls as well. Proteomic analysis was performed using mass spectrometry (MS) and targeted multiple reaction monitoring (MRM) and a large scale isobaric tags for relative and absolute quantitation (iTRAQ) approach. After the 16-week VLCD, there was a significant decrease in body weight and HbA1c in all patients, without differences between the two intervention groups. Targeted MRM analysis revealed differences in several proteins, which could be divided in diabetes-associated (fibrinogen, transthyretin), obesity-associated (complement C3), and diet-associated markers (apolipoproteins, especially apolipoprotein A-IV). To further investigate the effects of exercise, large scale iTRAQ analysis was performed. However, no proteins were found showing an exercise effect. Thus, in this study, specific proteins were found to be differentially expressed in type 2 diabetes patients versus controls and before and after a VLCD. These proteins are potential disease state and intervention specific biomarkers. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN76920690.