Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila.
Bidirectional axonal transport driven by kinesin and dynein along microtubules is critical to neuronal viability and function. To evaluate axonal transport mechanisms, we developed a high-resolution imaging system to track the movement of amyloid precursor protein (APP) vesicles in Drosophila segmental nerve axons. Computational analyses of a large number of moving vesicles in defined genetic backgrounds with partial reduction or overexpression of motor proteins enabled us to test with high precision existing and new models of motor activity and coordination in vivo. We discovered several previously unknown features of vesicle movement, including a surprising dependence of anterograde APP vesicle movement velocity on the amount of kinesin-1. This finding is largely incompatible with the biophysical properties of kinesin-1 derived from in vitro analyses. Our data also suggest kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and their direction and velocity are controlled at least in part by dynein intermediate chain.


An automated real-time microfluidic platform to probe single NK cell heterogeneity and cytotoxicity on-chip.
Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells. While conventional cytotoxicity assays rely on averaged end-point measures, crucial insights on the dynamics and heterogeneity of effector and target cell interactions cannot be extracted, emphasizing the need for dynamic single-cell analysis. Here, we present a fully automated droplet-based microfluidic platform that allowed the real-time monitoring of effector-target cell interactions and killing, allowing the screening of over 60,000 droplets identifying 2000 individual cellular interactions monitored over 10 h. During the course of incubation, we observed that the dynamics of cytotoxicity within the Natural Killer (NK) cell population varies significantly over the time. Around 20% of the total NK cells in droplets showed positive cytotoxicity against paired K562 cells, most of which was exhibited within first 4 h of cellular interaction. Using our single cell analysis platform, we demonstrated that the population of NK cells is composed of individual cells with different strength in their effector functions, a behavior masked in conventional studies. Moreover, the versatility of our platform will allow the dynamic and resolved study of interactions between immune cell types and the finding and characterization of functional sub-populations, opening novel ways towards both fundamental and translational research.


A retrospective study of paroxysmal nocturnal hemoglobinuria in pediatric and adolescent patients.
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease, especially in children, characterized by intravascular hemolysis, thrombotic events, serious infections and bone marrow failure. We describe 16 patients who were diagnosed with PNH in childhood or adolescence. The time interval between the onset of symptoms and the PNH diagnosis and its treatment were compared in patients with classic PNH versus PNH associated with bone marrow disorder (PNH/BMD). A greater delay in diagnosis was observed in classic PNH compared to PNH/BMD patients. The first group of patients had higher levels of LDH, total bilirubin and absolute reticulocyte count and a bigger PNH clone size compared to PNH/BMD patients; also thrombotic events were observed only in the classic form of PNH. Conversely, PNH/BMD patients showed lower median levels of platelets. Apart from standard supportive measures, four patients with classic PNH received eculizumab whereas four patients with PNH/BMD underwent hematopoietic stem cell transplantation. Our series confirm that the most frequent presentation of PNH in the pediatric-adolescent age is PNH/BMD. The delay between the onset of symptoms and PNH diagnosis is relevant principally in the classic form. Moreover, our study showed that any case of unexpected thrombosis represents a criterium to perform a PNH screening.


Real-time experiment interface for biological control applications.
The Real-time Experiment Interface (RTXI) is a fast and versatile real-time biological experimentation system based on Real-Time Linux. RTXI is open source and free, can be used with an extensive range of experimentation hardware, and can be run on Linux or Windows computers (when using the Live CD). RTXI is currently used extensively for two experiment types: dynamic patch clamp and closed-loop stimulation pattern control in neural and cardiac single cell electrophysiology. RTXI includes standard plug-ins for implementing commonly used electrophysiology protocols with synchronized stimulation, event detection, and online analysis. These and other user-contributed plug-ins can be found on the website (http://www.rtxi.org).


Developmental regulation of trigeminal TRPA1 by the cornea.
The cornea is densely innervated with nociceptive nerves that detect deleterious stimuli at the ocular surface and transduce these stimuli as sensations of pain. Thus, nociception is a major factor involved in preventing damage to corneal tissues. One class of molecules that is thought to be involved in detecting such stimuli is the transient receptor potential (TRP) family of ion channels. However, little is known about the acquisition of these channels during corneal development. Therefore, the present study examined the developmental acquisition of these receptors and elucidated certain parameters involved in this acquisition. Quantitative RT-PCR was used to measure the expression of genes including TRPA and Ret in vivo. In vitro cocultures between cornea and the ophthalmic lobe of the trigeminal ganglion were used to test interactions between nerves and corneas along with recombinant proteins. TRPA1 mRNA showed a progressive temporal increase in the ophthalmic lobe of the trigeminal ganglion in vivo during embryonic development. In vitro, TRPA1 expression was significantly increased in the ganglion when cocultured with cornea, compared to ganglia cultured alone. Similarly, the addition of exogenous neurotrophin-3 (NT3) protein to cultured ganglia increased the expression of TRPA1 more than 100-fold. Addition of NT3 and neurturin synergistically increased TRPA1 expression in embryonic day (E)8 ganglia, but this effect was lost at E12. At E8, Ret+ nonpeptidergic neurons are specified in the trigeminal ganglion. Corneal-derived factors increase TRPA1 expression in trigeminal nonpeptidergic neurons during their embryonic specification.


Results of the American Association of Bioanalysts national proficiency testing programme in andrology.
Proficiency testing samples for antisperm antibodies (ASAB), sperm count, morphology and vitality were mailed to participating laboratories. The majority participating utilized Immunobead ASAB procedures (81 versus 14% mixed antiglobulin reaction and 5% 'other'), and there was 95.6 +/- 1.2% agreement on the presence or absence of ASAB. The majority of laboratories utilized manual (79%) versus computer assisted semen analysis (CASA; 15%) methods. Approximately 64% used the haemocytometer and 26% used the Makler counting chambers for manual counts. Coefficients of variation (CV) in sperm counts ranged from 24 to 138%, with CASA displaying lower overall CV (53 +/- 8%) than manual methods (80 +/- 9%). A wide variation in the reports of percent normal morphology was noted (CVs calculated from arc sin transformed means ranged from 15 to 93%). Participants using American Society of Clinical Pathologists (ASCP) criteria reported sperm morphology values that were clustered in the 'normal' range (11 out of 12 samples), while those using strict criteria were clustered in the 'abnormal' range (10 out of 12 samples). Good agreement was observed in sperm vitality (overall mean CV = 18%). These data highlight the urgent need for improvement in overall quality of andrology testing and indicate that practical proficiency testing programmes can be made available on a large scale.


Dose-effect meta-analysis for psychopharmacological interventions using randomised data.
The current practice in meta-analysis of the effects of psychopharmacological interventions ignors the administered dose or restricts the analysis in a dose range. This may introduce unnecessary uncertainty and heterogeneity. Methods have been developed to integrate the dose-effect models in meta-analysis. We describe the two-stage and the one-stage models to conduct a dose-effect meta-analysis using common or random effects methods. We illustrate the methods on a dataset of selective serotonin reuptake inhibitor antidepressants. The dataset comprises 60 randomised controlled trials. The dose-effect is measured on an odds ratio scale and is modelled using restricted cubic splines to detect departure from linearity. The estimated summary curve indicates that the probability of response increases up to 30 mg/day of fluoxetine-equivalent which results in reaching 50% probability to respond. Beyond 40 mg/day, no further increase in the response is observed. The one-stage model includes all studies, resulting in slightly less uncertainty than the two-stage model where only part of the data is analysed. The dose-effect meta-analysis enables clinicians to understand how the effect of a drug changes as a function of its dose. Such analysis should be conducted in practice using the one-stage model that incorporates evidence from all available studies.


Clean indoor air policies in Wisconsin workplaces.
To describe the nature and extent of workplace environmental tobacco smoke exposures in Wisconsin. Descriptive data and confidence intervals from the Current Population Survey tobacco supplements of 1995-1996 and 1998-1999 are presented. The percent of indoor workers working under a smoke-free policy increased slightly, from 62% in 1995-1996 to 65% in 1998-1999. Respondents with a college degree were more likely to work under a smoke-free policy than those with a high school education or less. Among respondents with a work policy in 1998-1999, a complete ban on smoking reduced any workplace exposure in the past 2 weeks (4%) compared to a partial ban (26%) or an unrestricted policy (30%). Wisconsin has seen a small increase in workplace policies that ban smoking in the workplace. These policies are more likely to protect workers of higher socioeconomic status and may increase health disparities in tobacco-related diseases in the future.


Diagnostic exome sequencing to elucidate the genetic basis of likely recessive disorders in consanguineous families.
Rare, atypical, and undiagnosed autosomal-recessive disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic genetic tests fail to establish a diagnosis in many cases. We employed exome sequencing to identify the underlying molecular defects in patients with unresolved but putatively autosomal-recessive disorders in consanguineous families and postulated that the pathogenic variants would reside within homozygous regions. Fifty consanguineous families participated in the study, with a wide spectrum of clinical phenotypes suggestive of autosomal-recessive inheritance, but with no definitive molecular diagnosis. DNA samples from the patient(s), unaffected sibling(s), and the parents were genotyped with a 720K SNP array. Exome sequencing and array CGH (comparative genomic hybridization) were then performed on one affected individual per family. High-confidence pathogenic variants were found in homozygosity in known disease-causing genes in 18 families (36%) (one by array CGH and 17 by exome sequencing), accounting for the clinical phenotype in whole or in part. In the remainder of the families, no causative variant in a known pathogenic gene was identified. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and can be used to establish more detailed causative links between mutant genotypes and clinical phenotypes.


Effect of different disinfectant methods on the initial microtensile bond strength of a self-etch adhesive to dentin.
The aim of this in vitro study was to evaluate the effect of different disinfection methods on the initial microtensile bond strength of a two-step, self-etch adhesive to dentin. Twenty mandibular molars were sectioned parallel to the occlusal plane to expose the mid-coronal dentin. All of the teeth were divided into four groups (n = 5 per group): (1) in group OZ, the dentin surfaces were exposed to ozone gas from the Ozonytron X delivery system (OzonyTron X-Bioozonix, Munich, Germany), (2) in group ND, the dentin surfaces were irradiated with an Nd:YAG laser (Pulsmaster 600 IQ, American Dental Technologies, U.S.), (3) in group CHX, the dentin surfaces were treated with a 2% chlorhexidine solution, and (4) in the control group, no treatment was applied. In all of the groups, the teeth were restored with Clearfil SE Bond (Kuraray, Tokyo, Japan) and Clearfil Majesty Posterior (Kuraray, Tokyo, Japan), according to the manufacturer's instructions. The teeth were sectioned perpendicular to the bonded surface (surface area of approximately 1 mm(2)). Thus, six to seven specimens were obtained from each tooth, and a total of 34 specimens were analyzed in each group. The specimens were attached to the microtensile test machine (Micro Tensile Tester, T-61010 K, Bisco, U.S.). The data was analyzed using the one-way analysis of variance (ANOVA) and Tukey test (p < 0.05). Fracture modes of each specimen were determined using a stereomicroscope (SZ-PT Olympus, Tokyo, Japan) and a scanning electron microscope (SEM). The lowest bond strength occurred in the OZ group. Significant differences were determined only between group OZ and the other groups (group ND, group CHX, and control group) (p < 0.05). In conclusion, although ozone decreased the microtensile bond strength of the self-etch adhesive system to dentin, the Nd:YAG laser and 2% chlorhexidine did not change the microtensile bond strength so in context of the present study it would appear that the Nd:YAG laser and 2% chlorhexidine may be used as pre-restorative sterilization procedures on the dentin prior to the application of a two-step, self-etch adhesive.


Imaging of vibrating objects using speckle subtraction.
A simple method for imaging vibrational motion is proposed. The process consists of capturing two speckled images of a region illuminated by coherent radiation. One of the images is captured before the onset of motion and the other during motion. If the mean speckle intensity is below the threshold for detection or above the saturation intensity of the detector, subtracting the two images produces a high contrast image of the moving region. A theory is shown to agree well with experimental data.


A post-developmental genetic screen for zebrafish models of inherited liver disease.
Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such as simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecular pathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence and incidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD. To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a large-scale mutant screen, and adopted a whole genome sequencing approach for rapid identification of mutated genes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From 250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization), we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosis with no developmental defects. Histological analysis of mutants revealed several specific phenotypes, including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acute hepatocellular necrosis. This work has identified multiple post-developmental mutants and establishes zebrafish as a novel animal model for post-developmental inherited liver disease.


Six-month changes in spirituality and religiousness in alcoholics predict drinking outcomes at nine months.
Although spiritual change is hypothesized to contribute to recovery from alcohol dependence, few studies have used prospective data to investigate this hypothesis. Prior studies have also been limited to treatment-seeking and Alcoholics Anonymous (AA) samples. This study included alcohol-dependent individuals, both in treatment and not, to investigate the effect of spiritual and religious (SR) change on subsequent drinking outcomes, independent of AA involvement. Alcoholics (N = 364) were recruited for a panel study from two abstinence-based treatment centers, a moderation drinking program, and untreated individuals from the local community. Quantitative measures of SR change between baseline and 6 months were used to predict 9-month drinking outcomes, controlling for baseline drinking and AA involvement. Significant 6-month changes in 8 of 12 SR measures were found, which included private SR practices, beliefs, daily spiritual experiences, three measures of forgiveness, negative religious coping, and purpose in life. Increases in private SR practices and forgiveness of self were the strongest predictors of improvements in drinking outcomes. Changes in daily spiritual experiences, purpose in life, a general measure of forgiveness, and negative religious coping also predicted favorable drinking outcomes. SR change predicted good drinking outcomes in alcoholics, even when controlling for AA involvement. SR variables, broadly defined, deserve attention in fostering change even among those who do not affiliate with AA or religious institutions. Last, future research should include SR variables, particularly various types of forgiveness, given the strong effects found for forgiveness of self.


Totally robotic Roux-en-Y gastric bypass.
We hypothesized that we could develop a safe and effective technique for performing a totally robotic laparoscopic Roux-en-Y gastric bypass procedure using the da Vinci surgical system. We anticipated that the learning curve for this totally robotic procedure could be shorter than the learning curve for standard laparoscopic bariatric surgery. Retrospective case comparison study. Academic tertiary care center. Consecutive samples of patients who met National Institutes of Health (NIH) criteria for morbid obesity and who completed the Stanford Bariatric Surgery Program evaluation process. A port placement and robot positioning scheme was developed so that the entire case could be performed robotically. The first 10 patients who underwent a totally robotic laparoscopic Roux-en-Y gastric bypass were compared with a retrospective sample of 10 patients who had undergone laparoscopic Roux-en-Y gastric bypass surgery. Patient age, gender, body mass index (BMI), numbers of NIH-defined comorbidities, operative time, length of stay, and complications. No significant differences existed between the 2 patient series with regard to age, gender, or BMI. The median surgical times were significantly lower for the robotic procedures (169 vs 208 minutes; P = .03), as was the ratio of procedure time to BMI (3.8 vs 5.0 minutes per BMI for the laparoscopic cases; P = .04). This study details the first report, to our knowledge, of a totally robotic laparoscopic Roux-en-Y gastric bypass and demonstrates the feasibility, safety, and potential superiority of such a procedure. In addition, the learning curve may be significantly shorter with the robotic procedure. Further experience is needed to understand the long-term advantages and disadvantages of the totally robotic approach.


Solid tumor proteome and phosphoproteome analysis by high resolution mass spectrometry.
Kinases play a prominent role in tumor development, pointing to the presence of specific phosphorylation patterns in tumor tissues. Here, we investigate whether recently developed high resolution mass spectrometric (MS) methods for proteome and phosphoproteome analysis can also be applied to solid tumors. As tumor model, we used TG3 mutant mice carrying skin melanomas. At total of 100 microg of solid tumor lysate yielded a melanoma proteome of 4443 identified proteins, including at least 88 putative melanoma markers previously found by cDNA microarray technology. Analysis of 2 mg of lysate from dissected melanoma with titansphere chromatography and 8 mg with strong cation exchange together resulted in the identification of more than 5600 phosphorylation sites on 2250 proteins. The phosphoproteome included many hits from pathways important in melanoma. One-month storage at -80 degrees C did not significantly decrease the number of identified phosphorylation sites. Thus, solid tumor can be analyzed by MS-based proteomics with similar efficiency as cell culture models and in amounts compatible with biopsies.


Paediatric acute respiratory distress syndrome incidence and epidemiology (PARDIE): an international, observational study.
Paediatric acute respiratory distress syndrome (PARDS) is associated with high mortality in children, but until recently no paediatric-specific diagnostic criteria existed. The Pediatric Acute Lung Injury Consensus Conference (PALICC) definition was developed to overcome limitations of the Berlin definition, which was designed and validated for adults. We aimed to determine the incidence and outcomes of children who meet the PALICC definition of PARDS. In this international, prospective, cross-sectional, observational study, 145 paediatric intensive care units (PICUs) from 27 countries were recruited, and over a continuous 5 day period across 10 weeks all patients were screened for enrolment. Patients were included if they had a new diagnosis of PARDS that met PALICC criteria during the study week. Exclusion criteria included meeting PARDS criteria more than 24 h before screening, cyanotic heart disease, active perinatal lung disease, and preparation or recovery from a cardiac intervention. Data were collected on the PICU characteristics, patient demographics, and elements of PARDS (ie, PARDS risk factors, hypoxaemia severity metrics, type of ventilation), comorbidities, chest imaging, arterial blood gas measurements, and pulse oximetry. The primary outcome was PICU mortality. Secondary outcomes included 90 day mortality, duration of invasive mechanical and non-invasive ventilation, and cause of death. Between May 9, 2016, and June 16, 2017, during the 10 study weeks, 23 280 patients were admitted to participating PICUs, of whom 744 (3·2%) were identified as having PARDS. 95% (708 of 744) of patients had complete data for analysis, with 17% (121 of 708; 95% CI 14-20) mortality, whereas only 32% (230 of 708) of patients met Berlin criteria with 27% (61 of 230) mortality. Based on hypoxaemia severity at PARDS diagnosis, mortality was similar among those who were non-invasively ventilated and with mild or moderate PARDS (10-15%), but higher for those with severe PARDS (33% [54 of 165; 95% CI 26-41]). 50% (80 of 160) of non-invasively ventilated patients with PARDS were subsequently intubated, with 25% (20 of 80; 95% CI 16-36) mortality. By use of PALICC PARDS definition, severity of PARDS at 6 h after initial diagnosis (area under the curve [AUC] 0·69, 95% CI 0·62-0·76) discriminates PICU mortality better than severity at PARDS diagnosis (AUC 0·64, 0·58-0·71), and outperforms Berlin severity groups at 6 h (0·64, 0·58-0·70; p=0·01). The PALICC definition identified more children as having PARDS than the Berlin definition, and PALICC PARDS severity groupings improved the stratification of mortality risk, particularly when applied 6 h after PARDS diagnosis. The PALICC PARDS framework should be considered for use in future epidemiological and therapeutic research among children with PARDS. University of Southern California Clinical Translational Science Institute, Sainte Justine Children's Hospital, University of Montreal, Canada, Réseau en Santé Respiratoire du Fonds de Recherche Quebec-Santé, and Children's Hospital Los Angeles, Department of Anesthesiology and Critical Care Medicine.


Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol.
The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co-C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5'-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg(2+) and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5'-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H(2)S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.


Distribution of the intermediate elements operating in ER to Golgi transport.
We have used a 58 kDa membrane protein (p58) as a marker to study the transport pathway between the rough endoplasmic reticulum (ER) and the Golgi apparatus. Immunolocalization of p58 in fibroblasts showed its presence in a single cisterna and in small tubular and vesicular elements at the cis side of the Golgi apparatus. In addition, the protein was detected in large (200-500 nm in diameter) tubulovesicular structures, clustered in the Golgi region but also found in peripheral locations. These represent intermediates in ER to Golgi transport since they contained newly synthesized viral glycoproteins, arrested in cells at 15 degrees C. The peripheral structures accumulated at low temperature but reclustered rapidly to the Golgi region upon shift of cells back to 37 degrees C. This movement involved long intracellular distances and was efficiently inhibited by nocodazole, indicating that it requires the integrity of microtubules. In contrast, reclustering was unaffected by brefeldin A (BFA), suggesting that this compound affects ER to Golgi transport prior to the temperature-sensitive step. In BFA-treated cells p58 was localized to scattered, tubular, smooth ER clusters, found in close association with rough ER cisternae. The cellular distribution of the intermediate elements indicates that the sites of protein exit are widely distributed within the rough ER network. We suggest that the smooth ER locations where p58 accumulates in BFA-treated cells could represent such peripheral exit sites.


Multifaceted role of cycling DOF factor 3 (CDF3) in the regulation of flowering time and abiotic stress responses in Arabidopsis.
DNA-binding with one finger (DOF)-type transcription factors are involved in many fundamental processes in higher plants, from responses to light and phytohormones to flowering time and seed maturation, but their relation with abiotic stress tolerance is largely unknown. Here, we identify the roles of CDF3, an Arabidopsis DOF gene in abiotic stress responses and developmental processes like flowering time. CDF3 is highly induced by drought, extreme temperatures and abscisic acid treatment. The CDF3 T-DNA insertion mutant cdf3-1 is much more sensitive to drought and low temperature stress, whereas CDF3 overexpression enhances the tolerance of transgenic plants to drought, cold and osmotic stress and promotes late flowering. Transcriptome analysis revealed that CDF3 regulates a set of genes involved in cellular osmoprotection and oxidative stress, including the stress tolerance transcription factors CBFs, DREB2A and ZAT12, which involve both gigantea-dependent and independent pathways. Consistently, metabolite profiling disclosed that the total amount of some protective metabolites including γ-aminobutyric acid, proline, glutamine and sucrose were higher in CDF3-overexpressing plants. Taken together, these results indicate that CDF3 plays a multifaceted role acting on both flowering time and abiotic stress tolerance, in part by controlling the CBF/DREB2A-CRT/DRE and ZAT10/12 modules.


Evaluation of the Social Motivation Hypothesis of Autism: A Systematic Review and Meta-analysis.
The social motivation hypothesis posits that individuals with autism spectrum disorder (ASD) find social stimuli less rewarding than do people with neurotypical activity. However, functional magnetic resonance imaging (fMRI) studies of reward processing have yielded mixed results. To examine whether individuals with ASD process rewarding stimuli differently than typically developing individuals (controls), whether differences are limited to social rewards, and whether contradictory findings in the literature might be due to sample characteristics. Articles were identified in PubMed, Embase, and PsycINFO from database inception until June 1, 2017. Functional MRI data from these articles were provided by most authors. Publications were included that provided brain activation contrasts between a sample with ASD and controls on a reward task, determined by multiple reviewer consensus. When fMRI data were not provided by authors, multiple reviewers extracted peak coordinates and effect sizes from articles to recreate statistical maps using seed-based d mapping software. Random-effects meta-analyses of responses to social, nonsocial, and restricted interest stimuli, as well as all of these domains together, were performed. Secondary analyses included meta-analyses of wanting and liking, meta-regression with age, and correlations with ASD severity. All procedures were conducted in accordance with Meta-analysis of Observational Studies in Epidemiology guidelines. Brain activation differences between groups with ASD and typically developing controls while processing rewards. All analyses except the domain-general meta-analysis were planned before data collection. The meta-analysis included 13 studies (30 total fMRI contrasts) from 259 individuals with ASD and 246 controls. Autism spectrum disorder was associated with aberrant processing of both social and nonsocial rewards in striatal regions and increased activation in response to restricted interests (social reward, caudate cluster: d = -0.25 [95% CI, -0.41 to -0.08]; nonsocial reward, caudate and anterior cingulate cluster: d = -0.22 [95% CI, -0.42 to -0.02]; restricted interests, caudate and nucleus accumbens cluster: d = 0.42 [95% CI, 0.07 to 0.78]). Individuals with ASD show atypical processing of social and nonsocial rewards. Findings support a broader interpretation of the social motivation hypothesis of ASD whereby general atypical reward processing encompasses social reward, nonsocial reward, and perhaps restricted interests. This meta-analysis also suggests that prior mixed results could be driven by sample age differences, warranting further study of the developmental trajectory for reward processing in ASD.


Comparative genomics of Campylobacter concisus: Analysis of clinical strains reveals genome diversity and pathogenic potential.
In recent years, an increasing number of Campylobacter species have been associated with human gastrointestinal (GI) diseases including gastroenteritis, inflammatory bowel disease, and colorectal cancer. Campylobacter concisus, an oral commensal historically linked to gingivitis and periodontitis, has been increasingly detected in the lower GI tract. In the present study, we generated robust genome sequence data from C. concisus strains and undertook a comprehensive pangenome assessment to identify C. concisus virulence properties and to explain potential adaptations acquired while residing in specific ecological niche(s) of the GI tract. Genomes of 53 new C. concisus strains were sequenced, assembled, and annotated including 36 strains from gastroenteritis patients, 13 strains from Crohn's disease patients and four strains from colitis patients (three collagenous colitis and one lymphocytic colitis). When compared with previous published sequences, strains clustered into two main groups/genomospecies (GS) with phylogenetic clustering explained neither by disease phenotype nor sample location. Paired oral/faecal isolates, from the same patient, indicated that there are few genetic differences between oral and gut isolates which suggests that gut isolates most likely reflect oral strain relocation. Type IV and VI secretion systems genes, genes known to be important for pathogenicity in the Campylobacter genus, were present in the genomes assemblies, with 82% containing Type VI secretion system genes. Our findings indicate that C. concisus strains are genetically diverse, and the variability in bacterial secretion system content may play an important role in their virulence potential.


Role of C1858T Polymorphism of Lymphoid Tyrosine Phosphatase in Egyptian Children and Adolescents with Type 1 Diabetes.
Type 1 Diabetes Mellitus (T1DM) is a multifactorial autoimmune disease. The Protein Tyrosine Phosphatase Non-receptor 22 (PTPN22) gene is an important negative regulator of signal transduction through the T-cell Receptors (TCR). A PTPN22 polymorphism, C1858T, has been found to be a risk determinant for several autoimmune diseases, including T1DM, in different populations. The present study was aimed to analyze a possible association between the C1858T polymorphism in Egyptian children with T1DM. This case-control study included 240 children divided evenly between T1DM patients and controls. The PTPN22 C1858T polymorphism was genotyped using polymerase chain reaction with Restriction Fragment Length Polymorphism (RFLP). Both the 1858CΤ and 1858ΤΤ genotypes and the 1858T allele were found more frequently in patients (32.5% and 18.7%, respectively) than in controls (10% and 5.0%, respectively), P=0.013 and P=0.007, respectively. Among females, the 1858T allele was more common in patients (18%) than in controls (2.6%), P=0.014. These findings suggest that the PTPN22 1858T allele could be a T1DM susceptibility factor in the Egyptian population and that it might play a different role in susceptibility to T1DM according to gender in T1DM patients.


Stem cells and their niches.
A constellation of intrinsic and extrinsic cellular mechanisms regulates the balance of self-renewal and differentiation in all stem cells. Stem cells, their progeny, and elements of their microenvironment make up an anatomical structure that coordinates normal homeostatic production of functional mature cells. Here we discuss the stem cell niche concept, highlight recent progress, and identify important unanswered questions. We focus on three mammalian stem cell systems where large numbers of mature cells must be continuously produced throughout adult life: intestinal epithelium, epidermal structures, and bone marrow.


Highly luminescent terpyridinyl-ethynyl functionalized dithieno[3,2-b:2',3'-d]phospholes: synthesis, properties and complexation behavior.
The synthesis and advanced characterization, as well as the complexation behavior of a series of terpyridinyl-ethynyl substituted dithieno[3,2-b:2',3'-d]phospholes is reported. The functionalization of the dithienophosphole core furnishes the system with suitable ligand properties for a variety of transition metal centers (Zn(2+), Ru(2+), Pt(2+)) to form the corresponding complexes. The terpyridinyl-extended ligand systems show pronounced photoluminescence features that are beneficial for the formation of strongly luminescent transition metal complexes potentially to be employed in organic light emitting electrochemical cells. Theoretical calculations support the strong luminescence of the ligands resulting from pi-pi* transitions involving the extended pi-conjugated scaffold. The ligands were found to exhibit remarkable luminescence stability towards protonation, whereas complexation to transition metals shows distinct changes in the photophysical properties that depend of the nature of the transition metal employed.


Longitudinal analysis of change in individual-level needle and syringe coverage amongst a cohort of people who inject drugs in Melbourne, Australia.
Needle and syringe program (NSP) coverage is often calculated at the individual level. This method relates sterile needle and syringe acquisition to injecting frequency, resulting in a percentage of injecting episodes that utilise a sterile syringe. Most previous research using this method was restricted by their cross-sectional design, calling for longitudinal exploration of coverage. We used the data of 518 participants from an ongoing cohort of people who inject drugs in Melbourne, Australia. We calculated individual-level syringe coverage for the two weeks prior to each interview, then dichotomised the outcome as either "sufficient" (≥100% of injecting episodes covered by at least one reported sterile syringe) or "insufficient" (<100%). Time-variant predictors of change in recent coverage (from sufficient to insufficient coverage) were estimated longitudinally using logistic regression with fixed effects for each participant. Transitioning to methamphetamine injection (AOR:2.16, p=0.004) and a newly positive HCV RNA test result (AOR:4.93, p=0.001) were both associated with increased odds of change to insufficient coverage, whilst change to utilising NSPs as the primary source of syringe acquisition (AOR: 0.41, p=0.003) and opioid substitution therapy (OST) enrolment (AOR:0.51, p=0.013) were protective against a change to insufficient coverage. We statistically tested the transitions between time-variant exposure sub-groups and transitions in individual-level syringe coverage. Our results give important insights into means of improving coverage at the individual level, suggesting that methamphetamine injectors should be targeted, whilst both OST prescription and NSP should be expanded.


Reduced susceptibility of Staphylococcus aureus to vancomycin and platelet microbicidal protein correlates with defective autolysis and loss of accessory gene regulator (agr) function.
Loss of agr function, vancomycin exposure, and abnormal autolysis have been linked with both development of the GISA phenotype and low-level resistance in vitro to thrombin-induced platelet microbicidal proteins (tPMPs). We examined the potential in vitro interrelationships among these parameters in well-characterized, isogenic laboratory-derived and clinical Staphylococcus aureus isolates. The laboratory-derived S. aureus strains included RN6607 (agrII-positive parent) and RN6607V (vancomycin-passaged variant; hetero-GISA), RN9120 (RN6607 agr::tetM; agr II knockout parent), RN9120V (vancomycin-passaged variant), and RN9120-GISA (vancomycin passaged, GISA). Two serial isolates from a vancomycin-treated patient with recalcitrant, methicillin-resistant S. aureus (MRSA) endocarditis were also studied: A5937 (agrII-positive initial isolate) and A5940 (agrII-defective/hetero-GISA isolate obtained after prolonged vancomycin administration). In vitro tPMP susceptibility phenotypes were assessed after exposure of strains to either 1 or 2 mug/ml. Triton X-100- and vancomycin-induced lysis profiles were determined spectrophotometrically. For agrII-intact strain RN6607, vancomycin exposure in vitro was associated with modest increases in vancomycin MICs and reduced killing by tPMP, but no change in lysis profiles. In contrast, vancomycin exposure of agrII-negative RN9120 yielded a hetero-GISA phenotype and was associated with defects in lysis and reduced in vitro killing by tPMP. In the clinical isolates, loss of agrII function during prolonged vancomycin therapy was accompanied by emergence of the hetero-GISA phenotype and reduced tPMP killing, with no significant change in lysis profiles. An association was identified between loss of agrII function and the emergence of hetero-GISA phenotype during either in vitro or in vivo vancomycin exposure. In vitro, these events were associated with defective lysis and reduced susceptibility to tPMP. The precise mechanism(s) underlying these findings is the subject of current investigations.


Plasmodium protease ROM1 is important for proper formation of the parasitophorous vacuole.
Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV) where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(-) parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(-) sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(-) sporozoites do not differ in the rate of entry into Hepa1-6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(-) parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(-) parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host.


Life threatening illness and hospice care.
A retrospective study was undertaken of 25 families and their 26 ill children attending the first children's hospice in the United Kingdom. The study examined the family's perceptions of the care offered and the impact of chronic and life threatening illness. Eighteen (72%) of the families felt they had been well supported by the hospice and valued the family like atmosphere, perceiving the staff to be friendly, approachable, and helpful. The actual nature of hospice care, in an environment with other terminally ill children, was, however, considered a drawback for a few families. A number of families still had unmet needs, notably appropriate child minding when away from the hospice. The impact of chronic life threatening illness on the families was substantial. The parents (particularly the mothers), the index children, and their siblings all experienced much higher levels of psychological symptomatology than would have been expected from normal samples. While families felt greatly helped over symptom control, a proportion remained very worried about certain symptoms, particularly breathlessness, seizures, and pain. A high proportion of families were experiencing financial and employment difficulties as a result of their children's illnesses.


Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease.
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD.


Making the blastocyst: lessons from the mouse.
Mammalian preimplantation development, which is the period extending from fertilization to implantation, results in the formation of a blastocyst with three distinct cell lineages. Only one of these lineages, the epiblast, contributes to the embryo itself, while the other two lineages, the trophectoderm and the primitive endoderm, become extra-embryonic tissues. Significant gains have been made in our understanding of the major events of mouse preimplantation development, and recent discoveries have shed new light on the establishment of the three blastocyst lineages. What is less clear, however, is how closely human preimplantation development mimics that in the mouse. A greater understanding of the similarities and differences between mouse and human preimplantation development has implications for improving assisted reproductive technologies and for deriving human embryonic stem cells.


WE-G-217A-09: Phase Imaging Measurement of Static Magnetic Field Homogeneity.
The ACR MRI accreditation program requires measurement of the magnetic field homogeneity (MFH) in the annual QA. Full implementation of vendor methods can be very time consuming and incorporates techniques not available to clinical physicists. Conversely, many of the less involved techniques proposed tend to be less inaccurate and/or precise. Here, we propose a robust approach to MFH analysis using a simple phase mapping acquisition which is a time efficient compromise. The root mean square (r.m.s.) for MFH measurement is commonly used over multiple slices (∼24). In regions with high SNR, the MR signal can be reasonably assumed Gaussian distributed. Therefore, the standard deviation of phase values in ROI is equivalent to the r.m.s. of those. The standard deviations of phases in x, y and z are assumed uncorrelated. Thus, only axial, sagittal and coronal planes need to be acquired to approximate the MFH as opposed to a full 3D acquisition. To investigate this concept, phase images were acquired on four 1.5T clinical scanners and one 3.0 clinical scanner (MAGNETOM Espree, Siemens Medical Systems, Signa Excite HD 1.5T, and 3.0T GE Healthcare Technologies). The manufacturer phantoms were scanned using two echo times (delta_TE=TE2-TE1<5ms) using a simple 2D gradient echo acquisition to produce phase images. After acquiring phase difference images in three orthogonal planes, the standard deviation was calculated in three circular ROIs (Diameter=10, 20 and 30cm) in each plane, respectively, to estimate the MFH for the effective DSV. The MFH values in five scanners using this method were within vendor specifications for the DSV. Additionally, the measured MFH values compared favorably with vendor planned maintenance records with <0.1 ppm discrepancy. This proposed method may be a reliable and practical for regular MFH measurement in QA programs and providing an independent check of the vendor measurement.


Who pays for obesity?
Adult obesity is a growing problem. From 1962 to 2006, obesity prevalence nearly tripled to 35.1 percent of adults. The rising prevalence of obesity is not limited to a particular socioeconomic group and is not unique to the United States. Should this widespread obesity epidemic be a cause for alarm? From a personal health perspective, the answer is an emphatic "yes." But when it comes to justifications of public policy for reducing obesity, the analysis becomes more complex. A common starting point is the assertion that those who are obese impose higher health costs on the rest of the population—a statement which is then taken to justify public policy interventions. But the question of who pays for obesity is an empirical one, and it involves analysis of how obese people fare in labor markets and health insurance markets. We will argue that the existing literature on these topics suggests that obese people on average do bear the costs and benefits of their eating and exercise habits. We begin by estimating the lifetime costs of obesity. We then discuss the extent to which private health insurance pools together obese and thin, whether health insurance causes obesity, and whether being fat might actually cause positive externalities for those who are not obese. If public policy to reduce obesity is not justified on the grounds of external costs imposed on others, then the remaining potential justification would need to be on the basis of helping people to address problems of ignorance or self-control that lead to obesity. In the conclusion, we offer a few thoughts about some complexities of such a justification.


Deep-time climate legacies affect origination rates of marine genera.
Biodiversity dynamics are shaped by a complex interplay between current conditions and historic legacy. The interaction of short- and long-term climate change may mask the true relationship of evolutionary responses to climate change if not specifically accounted for. These paleoclimate interactions have been demonstrated for extinction risk and biodiversity change, but their importance for origination dynamics remains untested. Here, we show that origination probability in marine fossil genera is strongly affected by paleoclimate interactions. Overall, origination probability increases by 27.8% [95% CI (27.4%, 28.3%)] when a short-term cooling adds to a long-term cooling trend. This large effect is consistent through time and all studied groups. The mechanisms of the detected effect might be manifold but are likely connected to increased allopatric speciation with eustatic sea level drop caused by sustained global cooling. We tested this potential mechanism through which paleoclimate interactions can act on origination rates by additionally examining a proxy for habitat fragmentation. This proxy, continental fragmentation, has a similar effect on origination rates as paleoclimate interactions, supporting the importance of allopatric speciation through habitat fragmentation in the deep-time fossil record. The identified complex nature of paleoclimate interactions might explain contradictory conclusions on the relationship between temperature and origination in the previous literature. Our results highlight the need to account for complex interactions in evolutionary studies both between and among biotic and abiotic factors.


Temperamental differences between bipolar disorder, borderline personality disorder, and attention deficit/hyperactivity disorder: some implications for their diagnostic validity.
The relationship between borderline personality disorder (BPD), bipolar disorder (BD), and attention deficit/hyperactivity disorder (ADHD) requires further elucidation. Seventy-four adult psychiatric in- and out-patients, each of them having received one of these diagnoses on clinical assessment, were interviewed and compared in terms of diagnostic overlap, age and sex distribution, comorbid substance, anxiety and eating disorders, and affective temperament. Diagnostic overlap within the three disorders was 54%. Comorbidity patterns and gender ratio did not differ. The disorders showed very similar levels of cyclothymia. Sample size was small and only a limited number of validators were tested. The similar extent of cyclothymic temperament suggests mood lability as a common denominator of BPD, BD, and ADHD.


The Rosenberg Self-Esteem Scale: a bifactor answer to a two-factor question?
Despite its long-standing and widespread use, disagreement remains regarding the structure of the Rosenberg Self-Esteem Scale (RSES). In particular, concern remains regarding the degree to which the scale assesses self-esteem as a unidimensional or multidimensional (positive and negative self-esteem) construct. Using a sample of 3,862 high school students in the United Kingdom, 4 models were tested: (a) a unidimensional model, (b) a correlated 2-factor model in which the 2 latent variables are represented by positive and negative self-esteem, (c) a hierarchical model, and (d) a bifactor model. The totality of results including item loadings, goodness-of-fit indexes, reliability estimates, and correlations with self-efficacy measures all supported the bifactor model, suggesting that the 2 hypothesized factors are better understood as "grouping" factors rather than as representative of latent constructs. Accordingly, this study supports the unidimensionality of the RSES and the scoring of all 10 items to produce a global self-esteem score.


A comparison of the Monti and Casale (spiral Monti) procedures.
We present our long-term followup and comparison of outcomes between the Monti and Casale (spiral Monti) procedures in a large group of children and young adults. A retrospective chart review was done, including all patients undergoing the Monti or Casale procedure at our institution with a minimum followup of 6 months. Age at surgery, the bowel segment used, stomal location, the number and type of revisions or endoscopic procedures required after channel creation, problems catheterizing and channel continence were documented, and a database was created. Of 188 patients identified with at least 6 months of followup 109 underwent a Monti procedure, while 79 underwent a Casale procedure. Patient age at surgery was 10 months to 31 years (mean 10.2 years). Mean followup was 43 months for the entire cohort, and 47.2 and 37.2 months for the Monti and Casale groups, respectively. A total of 43 open revisions were required in 36 patients (19.1%). Stomal revisions accounted for 18 procedures, while subfascial revisions accounted for 25 in 17 (9.0%) and 21 (11.2%) patients, respectively. A total of 21 endoscopic procedures requiring anesthesia were performed in 17 patients (9.0%). In the Monti group stomal revision was required in 11 patients (10.1%), while subfascial revisions were required in 9 (8.3%). In the Casale group stomal revision was required in 6 patients (7.6%), while subfascial revisions were required in 12 (15.2%). Of the channels 98% were completely continent at the stoma. In a large population of children and young adults we report durable and reliable long-term results with the Monti and Casale procedures, including continence at the stoma. The only significant difference noted between the 2 procedures was a higher incidence of subfascial revisions for umbilical stomas in each group. The need for subfascial revision is highest in spiral Monti channels placed in the umbilicus.


Comparison of Inoculation with the InoqulA and WASP Automated Systems with Manual Inoculation.
The quality of sample inoculation is critical for achieving an optimal yield of discrete colonies in both monomicrobial and polymicrobial samples to perform identification and antibiotic susceptibility testing. Consequently, we compared the performance between the InoqulA (BD Kiestra), the WASP (Copan), and manual inoculation methods. Defined mono- and polymicrobial samples of 4 bacterial species and cloudy urine specimens were inoculated on chromogenic agar by the InoqulA, the WASP, and manual methods. Images taken with ImagA (BD Kiestra) were analyzed with the VisionLab version 3.43 image analysis software to assess the quality of growth and to prevent subjective interpretation of the data. A 3- to 10-fold higher yield of discrete colonies was observed following automated inoculation with both the InoqulA and WASP systems than that with manual inoculation. The difference in performance between automated and manual inoculation was mainly observed at concentrations of >10(6) bacteria/ml. Inoculation with the InoqulA system allowed us to obtain significantly more discrete colonies than the WASP system at concentrations of >10(7) bacteria/ml. However, the level of difference observed was bacterial species dependent. Discrete colonies of bacteria present in 100- to 1,000-fold lower concentrations than the most concentrated populations in defined polymicrobial samples were not reproducibly recovered, even with the automated systems. The analysis of cloudy urine specimens showed that InoqulA inoculation provided a statistically significantly higher number of discrete colonies than that with WASP and manual inoculation. Consequently, the automated InoqulA inoculation greatly decreased the requirement for bacterial subculture and thus resulted in a significant reduction in the time to results, laboratory workload, and laboratory costs.


Social support and service use among homeless persons with serious mental illness.
It has been widely hypothesized that persons with greater social support use fewer health care services, although previous studies have shown variable results. This study examines the relationship between levels of social support and formal service use among clients entering 18 community treatment programs for homeless persons with serious mental illness as part of the ACCESS demonstration project of the U.S. Center for Mental Health Services. Baseline and follow-up data on 1,828 clients entering the ACCESS program were used to evaluate the relationship between individual client socio-demographic and clinical characteristics, seven measures of social support, and levels of formal service use in this population. Three measures of social support were positively related to the use of outpatient medical services and one each to the use of substance abuse services and the total days of service use. Six out of seven measures of social support were positively related to the receipt of multiple services. It appears that social support is most strongly associated with improved access to an array of different services--a very important need among this population.


TNFR2: The new Treg switch?
Three recent publications identified the TNF/TNR2 pathway as a new target to reduce graft-versus-host-disease through regulatory T cells activation or to potentially switch on a strong anti-leukemic effect through regulatory T cells blockade in allogeneic hematopoietic stem cell transplantation. This identified the TNF/TNR2 pathway as a swith and as a new target for immune checkpoint therapy to modulate the immune regulation in this clinical setting.


SU-E-T-446: Effect of Dose Calculation Grid Size Variability on the Specification of Spinal Cord Dose Tolerance for Spinal Stereotactic Body Radiation Therapy.
To investigate the effect of the dose grid resolution variability on the spinal cord dose for spinal SBRT treatments. 10 CyberKnife plans were selected for the proximity of the PTV to the spinal cord. All dose distributions were calculated with Monte Carlo using high spatial resolution and minimal relative uncertainty. The plans were renormalized to a 16 Gy prescription dose and to ensure a target coverage > 95% in order to compare the calculated dose distributions. Each dose matrix was resampled 12 times, covering a grid resolution range of 0.95 mm to 13.52 mm. The spinal cord DVHs were generated for each resampled dose grid. The variations of the maximum point dose (DmaxCord) and dose-coverage to partial volumes (D[V]) up to 5 cc were investigated against the grid resolution. The mean variation of DmaxCord with grid resolution is characterized by an inverse power law, with a sharp initial decrease leading to potentially large underestimates of DmaxCord (24%, 40% and 55% at resolutions of 2 mm, 4 mm and 8 mm). The variability of mean D[V] values decreases from smaller to larger grid resolutions, however large disparities are observed between patient plans. We introduced the variability threshold volume (Vth) as a constraint to express the dose coverage independently from the grid resolution. For resolutions up to 8 mm, the mean Vth value is (0.96±0.10) cc with a corresponding dose coverage of (26± 12) % relative to the initial DmaxCord value. Dose distributions calculated with grid resolutions larger than 2 mm could Result in significant underestimates of DmaxCord. Furthermore, the sensitivity of the dose coverage to grid resolution variability is patient dependent. Consequently, a specified cord dose tolerance should be quoted at a particular grid resolution uniformly adopted between institutions; 2 mm or less is an appropriate value.


New roles for the de-ubiquitylating enzyme OTUD4 in an RNA-protein network and RNA granules.
Mechanisms that regulate the formation of membrane-less cellular organelles, such as neuronal RNA granules and stress granules, have gained increasing attention over the past years. These granules consist of RNA and a plethora of RNA-binding proteins. Mutations in RNA-binding proteins have been found in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). By performing pulldown experiments and subsequent mass spectrometry on mouse brain lysates, we discovered that the de-ubiquitylating enzyme OTU domain-containing protein 4 (OTUD4) unexpectedly is part of a complex network of multiple RNA-binding proteins, including core stress granule factors, such as FMRP (also known as FMR1), SMN1, G3BP1 and TIA1. We show that OTUD4 binds RNA, and that several of its interactions with RNA-binding proteins are RNA dependent. OTUD4 is part of neuronal RNA transport granules in rat hippocampal neurons under physiological conditions, whereas upon cellular stress, OTUD4 is recruited to cytoplasmic stress granules. Knockdown of OTUD4 in HeLa cells resulted in defects in stress granule formation and led to apoptotic cell death. Together, we characterize OTUD4 as a new RNA-binding protein with a suggested function in regulation of translation.


Coping with perturbations to a layout somersault in tumbling.
Tumbling is a dynamic movement requiring control of the linear and angular momenta generated during the approach and takeoff phases. Both of these phases are subject to some variability even when the gymnast is trying to perform a given movement repeatedly. This paper used a simulation model of tumbling takeoff to establish how well gymnasts can cope with perturbations of the approach and takeoff phases. A five segment planar simulation model with torque generators at each joint was developed to simulate tumbling takeoffs. The model was customised to an elite gymnast by determining subject specific inertia and torque parameters and a simulation was produced which closely matched a performance of a layout somersault by the gymnast. The performance of a layout somersault was found to be sensitive to the approach characteristics and the activation timings but relatively insensitive to the elasticity of the track and maximum muscle strength. Appropriate variation of the activation timings used during the takeoff phase was capable of coping with moderate perturbations of the approach characteristics. A model of aerial movement established that variation of body configuration in the flight phase was capable of adjusting for takeoff perturbations that would lead to rotation errors of up to 8%. Providing the errors in perceiving approach characteristics are less than 5% or 5 degrees and the errors in timing activations are less than 7ms, perturbations in the approach can be accommodated using adjustments during takeoff and flight.


Subcellular nutrient element localization and enrichment in ecto- and arbuscular mycorrhizas of field-grown beech and ash trees indicate functional differences.
Mycorrhizas are the chief organ for plant mineral nutrient acquisition. In temperate, mixed forests, ash roots (Fraxinus excelsior) are colonized by arbuscular mycorrhizal fungi (AM) and beech roots (Fagus sylvatica) by ectomycorrhizal fungi (EcM). Knowledge on the functions of different mycorrhizal species that coexist in the same environment is scarce. The concentrations of nutrient elements in plant and fungal cells can inform on nutrient accessibility and interspecific differences of mycorrhizal life forms. Here, we hypothesized that mycorrhizal fungal species exhibit interspecific differences in mineral nutrient concentrations and that the differences correlate with the mineral nutrient concentrations of their associated root cells. Abundant mycorrhizal fungal species of mature beech and ash trees in a long-term undisturbed forest ecosystem were the EcM Lactarius subdulcis, Clavulina cristata and Cenococcum geophilum and the AM Glomus sp. Mineral nutrient subcellular localization and quantities of the mycorrhizas were analysed after non-aqueous sample preparation by electron dispersive X-ray transmission electron microscopy. Cenococcum geophilum contained the highest sulphur, Clavulina cristata the highest calcium levels, and Glomus, in which cations and P were generally high, exhibited the highest potassium levels. Lactarius subdulcis-associated root cells contained the highest phosphorus levels. The root cell concentrations of K, Mg and P were unrelated to those of the associated fungal structures, whereas S and Ca showed significant correlations between fungal and plant concentrations of those elements. Our results support profound interspecific differences for mineral nutrient acquisition among mycorrhizas formed by different fungal taxa. The lack of correlation between some plant and fungal nutrient element concentrations may reflect different retention of mineral nutrients in the fungal part of the symbiosis. High mineral concentrations, especially of potassium, in Glomus sp. suggest that the well-known influence of tree species on chemical soil properties may be related to their mycorrhizal associates.


An assessment of the fine sediment dynamics in an upland river system: INCA-Sed modifications and implications for fisheries.
There is a need for better links between hydrology and ecology, specifically between landscapes and riverscapes to understand how processes and factors controlling the transport and storage of environmental pollution have affected or will affect the freshwater biota. Here we show how the INCA modelling framework, specifically INCA-Sed (the Integrated Catchments model for Sediments) can be used to link sediment delivery from the landscape to sediment changes in-stream. INCA-Sed is a dynamic, process-based, daily time step model. The first complete description of the equations used in the INCA-Sed software (version 1.9.11) is presented. This is followed by an application of INCA-Sed made to the River Lugg (1077 km(2)) in Wales. Excess suspended sediment can negatively affect salmonid health. The Lugg has a large and potentially threatened population of both Atlantic salmon (Salmo salar) and Brown Trout (Salmo trutta). With the exception of the extreme sediment transport processes, the model satisfactorily simulated both the hydrology and the sediment dynamics in the catchment. Model results indicate that diffuse soil loss is the most important sediment generation process in the catchment. In the River Lugg, the mean annual Guideline Standard for suspended sediment concentration, proposed by UKTAG, of 25 mg l(-1) is only slightly exceeded during the simulation period (1995-2000), indicating only minimal effect on the Atlantic salmon population. However, the daily time step simulation of INCA-Sed also allows the investigation of the critical spawning period. It shows that the sediment may have a significant negative effect on the fish population in years with high sediment runoff. It is proposed that the fine settled particles probably do not affect the salmonid egg incubation process, though suspended particles may damage the gills of fish and make the area unfavourable for spawning if the conditions do not improve.


CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis.
Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5-/- cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5-/- cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.


Morphology of perivascular spaces and enclosed blood vessels in young to middle-aged healthy adults at 7T: Dependences on age, brain region, and breathing gas.
Perivascular spaces (PVSs) are fluid-filled spaces surrounding penetrating blood vessels in the brain and are an integral pathway of the glymphatic system. A PVS and the enclosed blood vessel are commonly visualized as a single vessel-like complex (denoted as PVSV) in high-resolution MRI images. Quantitative characterization of the PVSV morphology in MRI images in healthy subjects may serve as a reference for detecting disease related PVS and/or blood vessel alterations in patients with brain diseases. To this end, we evaluated the age dependences, spatial heterogeneities, and dynamic properties of PVSV morphological features in 45 healthy subjects (21-55 years old), using an ultra-high-resolution three-dimensional transverse relaxation time weighted MRI sequence (0.41 × 0.41 × 0.4 mm3) at 7T. Quantitative PVSV parameters, including apparent diameter, count, volume fraction (VF), and relative contrast to noise ratio (rCNR) were calculated in the white matter and subcortical structures. Dynamic changes were induced by carbogen breathing which are known to induce vasodilation and increase the blood oxygenation level in the brain. PVSV count and VF significantly increased with age in basal ganglia (BG), so did rCNR in BG, midbrain, and white matter (WM). Apparent PVSV diameter also showed a positive association with age in the three brain regions, although it did not reach statistical significance. The PVSV VF and count showed large inter-subject variations, with coefficients of variation ranging from 0.17 to 0.74 after regressing out age and gender effects. Both apparent diameter and VF exhibited significant spatial heterogeneity, which cannot be explained solely by radio-frequency field inhomogeneities. Carbogen breathing significantly increased VF in BG and WM, and rCNR in thalamus, BG, and WM compared to air breathing. Our results are consistent with gradual dilation of PVSs with age in healthy adults. The PVSV morphology exhibited spatial heterogeneity and large inter-subject variations and changed during carbogen breathing compared to air breathing.


Neurological deficit of the facial nerve after root canal treatment.
Here we report the unusual case of a patient who suffered neurological deficit of the facial nerve as a complication of a root canal treatment. During the canal treatment, 3% hydrogen peroxide and 90% ethanol rinses were performed. The present case demonstrated rinse toxicity to vital tissue as evidenced by severe clinical damage. Chlorhexidine can be used instead of potentially toxic rinses with good antimicrobiological results. Furthermore, special attention should be paid to determination of the root canal length and the integrity of the canal system before rinsing. Also irrigation should be applied at a low pressure.


A reliable and reproducible test for adequate glucose counterregulation in type I diabetes mellitus.
The safety, reproducibility, and reliability of an insulin infusion test for assessment of adequate glucose counterregulation were evaluated in 18 patients with type I (insulin-dependent) diabetes mellitus. When the test (a 60-min, 30-mU/m2/min insulin infusion) was administered on three separate occasions at 3-4-wk intervals, coefficients of variation for plasma glucose and counterregulatory hormone (glucagon, epinephrine, cortisol, and growth hormone) responses averaged less than 8%. No patient experienced symptoms requiring discontinuation of the test and plasma glucose concentrations increased spontaneously after stopping the insulin infusion. Using objective criteria based on plasma glucose nadirs or postnadir rates of plasma glucose recovery, no patient judged to have adequate glucose counterregulation by the test (postnadir rates of plasma glucose recovery or plasma glucose nadir above 0.4 mg/dl/min and 45 mg/dl) developed severe hypoglycemia (plasma glucose less than 40 mg/dl) during up to 7 mo of intensive insulin therapy, whereas nearly all patients with inadequate counterregulation did. We conclude that this test, when performed in standardized conditions, is safe and reproducible and can reliably predict those patients with type I diabetes who are at risk of developing severe hypoglycemia during intensive insulin therapy.


Genetic Analysis of Arrhythmogenic Diseases in the Era of NGS: The Complexity of Clinical Decision-Making in Brugada Syndrome.
The use of next-generation sequencing enables a rapid analysis of many genes associated with sudden cardiac death in diseases like Brugada Syndrome. Genetic variation is identified and associated with 30-35% of cases of Brugada Syndrome, with nearly 20-25% attributable to variants in SCN5A, meaning many cases remain undiagnosed genetically. To evaluate the role of genetic variants in arrhythmogenic diseases and the utility of next-generation sequencing, we applied this technology to resequence 28 main genes associated with arrhythmogenic disorders. A cohort of 45 clinically diagnosed Brugada Syndrome patients classified as SCN5A-negative was analyzed using next generation sequencing. Twenty-eight genes were resequenced: AKAP9, ANK2, CACNA1C, CACNB2, CASQ2, CAV3, DSC2, DSG2, DSP, GPD1L, HCN4, JUP, KCNE1, KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNQ1, NOS1AP, PKP2, RYR2, SCN1B, SCN3B, SCN4B, SCN5A, SNTA1, and TMEM43. A total of 85 clinically evaluated relatives were also genetically analyzed to ascertain familial segregation. Twenty-two patients carried 30 rare genetic variants in 12 genes, only 4 of which were previously associated with Brugada Syndrome. Neither insertion/deletion nor copy number variation were detected. We identified genetic variants in novel candidate genes potentially associated to Brugada Syndrome. These include: 4 genetic variations in AKAP9 including a de novo genetic variation in 3 positive cases; 5 genetic variations in ANK2 detected in 4 cases; variations in KCNJ2 together with CASQ2 in 1 case; genetic variations in RYR2, including a de novo genetic variation and desmosomal proteins encoding genes including DSG2, DSP and JUP, detected in 3 of the cases. Larger gene panels or whole exome sequencing should be considered to identify novel genes associated to Brugada Syndrome. However, application of approaches such as whole exome sequencing would difficult the interpretation for clinical purposes due to the large amount of data generated. The identification of these genetic variants opens new perspectives on the implications of genetic background in the arrhythmogenic substrate for research purposes. As a paradigm for other arrhythmogenic diseases and for unexplained sudden death, our data show that clinical genetic diagnosis is justified in a family perspective for confirmation of genetic causality. In the era of personalized medicine using high-throughput tools, clinical decision-making is increasingly complex.


Investigating the endemic transmission of the hepatitis C virus.
The hepatitis C virus (HCV) infects at least 3% of people worldwide and is a leading global cause of liver disease. Although HCV spread epidemically during the 20th century, particularly by blood transfusion, it has clearly been present in human populations for several centuries. Here we attempt to redress the paucity of investigation into how long-term endemic transmission of HCV has been maintained. Such transmission not only represents the 'natural' route of infection but also contributes to new infections today. As a first step, we investigate the hypothesis that HCV can be mechanically transmitted by biting arthropods. Firstly, we use a combined bioinformatic and geographic approach to build a spatial database of endemic HCV infection and demonstrate that this can be used to geographically compare endemic HCV with the range distributions of potential vector species. Second, we use models from mathematical epidemiology to investigate if the parameters that describe the biting behaviour of vectors are consistent with a proposed basic reproduction number (R0) for HCV, and with the sustained transmission of the virus by mechanical transmission. Our analyses indicate that the mechanical transmission of HCV is plausible and that much further research into endemic HCV is needed.


Early intervention in post-traumatic stress disorder without exposure to trauma memories using internet-delivered cognitive therapy: A pilot case series.
Trauma-focussed psychological interventions are the treatments of choice for post-traumatic stress disorder (PTSD). As many clinical services receive high demand for PTSD interventions, strategies to improve treatment efficiency are needed. Some people seek help in the early phase post-trauma, including as soon as the first few months. It is unclear whether all components of trauma-focussed CBT are needed in this initial stage. Providing brief intervention in this early phase without work on trauma memories may be feasible and effective. This service evaluation study describes a case series of five participants experiencing PTSD following recent traumas. Participants completed a shortened 6-week form of Internet-delivered Cognitive Therapy for PTSD (iCT-PTSD), which used fewer treatment modules and focussed primarily on psychoeducation about PTSD, and two key treatment components, 'reclaiming your life' and trigger discrimination. Unlike the full course of iCT-PTSD, this format did not include working directly with trauma memories. The intervention was associated with large reductions in symptoms of PTSD, depression and anxiety at the 6-week timepoint, which were maintained at 3-month follow-up. Scores on the composite PTSD measure showed an average reduction of 91% between baseline and end of follow-up. One client required an extension to the weekly phase of treatment and received further treatment modules. All were discharged after follow-up and did not require further treatment. The findings provide preliminary evidence that this briefer format of iCT-PTSD was of benefit for those seeking support following recent traumas. Further examination in a larger controlled study is required.


Beyond the plot: technology extrapolation domains for scaling out agronomic science.
Ensuring an adequate food supply in systems that protect environmental quality and conserve natural resources requires productive and resource-efficient cropping systems on existing farmland. Meeting this challenge will be difficult without a robust spatial framework that facilitates rapid evaluation and scaling-out of currently available and emerging technologies. Here we develop a global spatial framework to delineate 'technology extrapolation domains' based on key climate and soil factors that govern crop yields and yield stability in rainfed crop production. The proposed framework adequately represents the spatial pattern of crop yields and stability when evaluated over the data-rich US Corn Belt. It also facilitates evaluation of cropping system performance across continents, which can improve efficiency of agricultural research that seeks to intensify production on existing farmland. Populating this biophysical spatial framework with appropriate socio-economic attributes provides the potential to amplify the return on investments in agricultural research and development by improving the effectiveness of research prioritization and impact assessment.


Reducing wasting in young children with preventive supplementation: a cohort study in Niger.
To compare the incidence of wasting, stunting, and mortality among children aged 6 to 36 months who are receiving preventive supplementation with either ready-to-use supplementary foods (RUSFs) or ready-to-use therapeutic foods (RUTFs). Children aged 6 to 36 months in 12 villages of Maradi, Niger, (n = 1645) received a monthly distribution of RUSFs (247 kcal [3 spoons] per day) for 6 months or RUTFs (500-kcal sachet per day) for 4 months. We compared the incidence of wasting, stunting, and mortality among children who received preventive supplementation with RUSFs versus RUTFs. The effectiveness of RUSF supplementation depended on receipt of a previous preventive intervention. In villages in which a preventive supplementation program was previously implemented, the RUSF strategy was associated with a 46% (95% confidence interval [CI]: 6%-69%) and 59% (95% CI: 17%-80%) reduction in wasting and severe wasting, respectively. In contrast, in villages in which the previous intervention was not implemented, we found no difference in the incidence of wasting or severe wasting according to type of supplementation. Compared with the RUTF strategy, the RUSF strategy was associated with a 19% (95% CI: 0%-34%) reduction in stunting overall. We found that the relative performance of a 6-month RUSF supplementation strategy versus a 4-month RUTF strategy varied with receipt of a previous nutritional intervention. Contextual factors will continue to be important in determining the dose and duration of supplementation that will be most effective, acceptable, and sustainable for a given setting.


Immunogenicity and efficacy of codon optimized DNA vaccines encoding the F-protein of respiratory syncytial virus.
Respiratory syncytial virus F-protein (RSV-F) is poorly expressed from DNA expression plasmids containing the wild type RSV-F open reading frame. By codon optimization, premature polyadenylation signals were deleted and a striking enhancement of RSV-F expression levels was achieved. Therefore, the immunogenicity and efficacy of wild type DNA vaccines were compared to codon optimized expression plasmids encoding full-length RSV-F or its ectodomain. Mice were immunized twice with the different DNA vaccines followed by an RSV challenge. Only codon optimized DNA vaccines and in particular the one encoding the ectodomain of RSV-F induced substantial antibody levels and reduced viral load 13-170-fold. Thus, codon optimization enhances the immunogenicity and efficacy of RSV encoding DNA vaccines.


Establishing and Evaluating a Study Questionnaire on Knowledge and Attitudes of Healthcare Professionals Towards Recreational and Medical Cannabis Across Europe.
The present survey was a preliminary to a European research project on the attitude and knowledge of healthcare professionals towards the use of medical cannabis. The objective was to evaluate the readability, understandability, and relevance of a first version of the study questionnaire before preparing the finalized questionnaire, which will be subsequently proposed to European healthcare professionals on a large scale. A cross-sectional study was conducted between December 2019 and May 2020. We established an electronic evaluation questionnaire relating to the study questionnaire. This evaluation questionnaire was proposed to multidisciplinary experts from all over Europe. Feedback from the evaluation questionnaire was considered for improving and finalizing the study questionnaire. 66 evaluation questionnaires were collected from nine European countries (Cyprus, France, Germany, Italy, Lithuania, Portugal, Spain, Sweden, United Kingdom), which corresponded to a participation rate of 41.5%. Most participants were women (65.2%, n = 43). The mean age was 39.5 years ± 11.6. Each participant could specify several occupations. There were 25 pharmacologists, 24 physicians, ten pharmacists, four university teachers, three epidemiologists or public health experts, one nurse, one biotechnologist, one microbiologist, and one police researcher. Overall, 84.8% of participants were interested in the topic of the survey on the knowledge and attitudes of healthcare professionals towards recreational and medical cannabis across Europe. Participants were satisfied with all but six of the proposed questions. In addition, two additional questions were subject for comments despite a high level of satisfaction. Consequently, the concerned questions (n = 8) were modified. This evaluation survey was a necessary step to improve the quality of the future research project. The positive feedback encourages the authors to proceed with the project on a European scale, scheduled for 2021.


Newborn Individual Development Care and Assessment Program (NIDCAP): a systematic review of the literature.
Important advancements have been made in the care of preterm infants. Health services have introduced various methods aimed at promoting attachment, breastfeeding, and neurological development. The Newborn Individualized Developmental Care and Assessment Program (NIDCAP), developed to stimulate preterm infants at levels adapted to the child's degree of neurological maturity, is increasingly being used. The aim was to investigate the impact of NIDCAP on the psychomotor development, neurological status, medical/nursing care outcomes, and parental perceptions. A further aim was to evaluate the cost-related effects of NIDCAP. A literature search up to September 2007 was performed. The reviewed papers were assessed for methodological quality and only statistically significant findings were extracted. The evidence compiled on the effects of NIDCAP is based on 12 articles from six randomized controlled trials that included approximately 250 children. Each of the studies was assessed as having medium quality. Most of the studies were small and many investigated a huge number of outcome variables, which decreased their scientific strength. On outcome variables in which a significant difference was found between the intervention (NIDCAP) and control groups, most studies showed better results for the NIDCAP group. This was particularly valid for cognitive and psychomotor development. Four studies also showed a reduced need for respiratory support for the NIDCAP group. No studies were identified that weighed the total cost of NIDCAP against its effects. Despite promising findings, primarily on cognitive and motor development, the scientific evidence on the effects of NIDCAP is limited. Shortcomings in design and methods in the reviewed studies hamper far-reaching claims on the effectiveness of the method. Scientific grounds for assessing the effects of NIDCAP would be substantially enhanced by a sufficiently comprehensive study with extended follow-up and a clear focus on a few important outcome variables.


Dynamic evolution of topological defects around drops and bubbles rising in a nematic liquid crystal.
We report observations of topological defects around drops and bubbles that rise through a vertically aligned nematic liquid crystal. We provide direct evidence for downstream convection of the Saturn-ring defect and its transformation to a hyperbolic point defect. The point defect is convected further in the wake of the drop or bubble as the rising velocity increases. In equilibrium, both defect configurations may persist for long times in the narrow cell. But the point defect sometimes spontaneously opens into a Saturn ring, indicating the latter as the globally stable configuration in the presence of tight wall confinement.


Plasma Proteomics to Identify Drug Targets for Ischemic Heart Disease.
Integrated analyses of plasma proteomic and genetic markers in prospective studies can clarify the causal relevance of proteins and discover novel targets for ischemic heart disease (IHD) and other diseases. The purpose of this study was to examine associations of proteomics and genetics data with IHD in population studies to discover novel preventive treatments. We conducted a nested case-cohort study in the China Kadoorie Biobank (CKB) involving 1,971 incident IHD cases and 2,001 subcohort participants who were genotyped and free of prior cardiovascular disease. We measured 1,463 proteins in the stored baseline samples using the OLINK EXPLORE panel. Cox regression yielded adjusted HRs for IHD associated with individual proteins after accounting for multiple testing. Moreover, cis-protein quantitative loci (pQTLs) identified for proteins in genome-wide association studies of CKB and of UK Biobank were used as instrumental variables in separate 2-sample Mendelian randomization (MR) studies involving global CARDIOGRAM+C4D consortium (210,842 IHD cases and 1,378,170 controls). Overall 361 proteins were significantly associated at false discovery rate <0.05 with risk of IHD (349 positively, 12 inversely) in CKB, including N-terminal prohormone of brain natriuretic peptide and proprotein convertase subtilisin/kexin type 9. Of these 361 proteins, 212 had cis-pQTLs in CKB, and MR analyses of 198 variants in CARDIOGRAM+C4D identified 13 proteins that showed potentially causal associations with IHD. Independent MR analyses of 307 cis-pQTLs identified in Europeans replicated associations for 4 proteins (FURIN, proteinase-activated receptor-1, Asialoglycoprotein receptor-1, and matrix metalloproteinase-3). Further downstream analyses showed that FURIN, which is highly expressed in endothelial cells, is a potential novel target and matrix metalloproteinase-3 a potential repurposing target for IHD. Integrated analyses of proteomic and genetic data in Chinese and European adults provided causal support for FURIN and multiple other proteins as potential novel drug targets for treatment of IHD.


Exploring the transcriptome of the burrowing nematode Radopholus similis.
Radopholus similis is an important nematode pest on fruit crops in the tropics. Unraveling the transcriptome of this migratory plant-parasitic nematode can provide insight in the parasitism process and lead to more efficient control measures. For the first high throughput molecular characterization of this devastating nematode, 5,853 expressed sequence tags from a mixed stage population were generated. Adding 1,154 tags from the EST division of GenBank for subsequent analysis, resulted in a total of 7,007 ESTs, which represent approximately 3,200 genes. The mean G + C content of the nucleotides at the third codon position (GC3%) was calculated to be as high as 64.8%, the highest for nematodes reported to date. BLAST-searches resulted in about 70% of the clustered ESTs having homology to (DNA and protein) sequences from the GenBank database, whereas one-third of them did not match to any known sequence. Roughly 40% of these latter sequences are predicted to be coding, representing putative novel protein coding genes. Functional annotation of the sequences by GO annotation revealed the abundance of genes involved in reproduction and development, which reflects the nematode population biology. Genes with a role in the parasitism process are identified, as well as genes essential for nematode survival, providing information useful for parasite control. No evidence was found for the presence of trans-spliced leader sequences commonly occurring in nematodes, despite the use of various approaches. In conclusion, we found three different sources for the EST sequences: the majority has a nuclear origin, approximately 1% of the EST sequences are derived from the mitochondrial transcriptome, and interestingly, 1% of the tags are with high probability derived from Wolbachia, providing the first molecular indication for the presence of this endosymbiont in a plant-parasitic nematode.


Co-suspension delivery technology in pressurized metered-dose inhalers for multi-drug dosing in the treatment of respiratory diseases.
Technologies for long-term delivery of aerosol medications in asthma and chronic obstructive pulmonary disease have improved over the past 2 decades with advancements in our understanding of the physical chemistry of aerosol formulations, device engineering, aerosol physics, and pulmonary biology. However, substantial challenges remain when a patient is required to use multiple inhaler types, multiple medications, and/or combinations of medications. Combining multiple drugs into a single inhaler while retaining appropriate dosing of the individual agents in the combination may enhance patient adherence to therapy and reduce device errors that occur when patients are using multiple inhalers. Pressurized metered-dose inhaler (pMDI) devices are widely used by patients for acute symptom relief as well as maintenance treatment, so the pMDI may be a suitable option with which to explore medication combinations. However, optimizing drug formulation remains a key challenge for pMDI delivery systems. This article introduces a new pMDI formulation approach: co-suspension delivery technology, which uses drug crystals with porous, low-density phospholipid particles engineered to deliver combinations of drugs to the airways with accurate and consistent dosing via pMDIs, independent of medication types and combinations. We describe the key characteristics of pMDIs, and discuss the rationale for the co-suspension delivery technology platform based on the limitations associated with traditional formulations. Finally, we discuss the clinical implications of co-suspension delivery technology for developing combination drug therapies administered by pMDIs.


Resection of the second portion of the duodenum sacrificing the minor papilla but preserving the pancreas for a recurrent duodenal adenocarcinoma: report of a case.
Duodenal adenocarcinoma is a relatively rare malignancy and pancreaticoduodenectomy would be a standard procedure to achieve curative resection. We report a case of resection of the 2nd portion of the duodenum with nodal dissection preserving the pancreas. The patient was a 75-year-old man with right-sided paresis suffering from early cancer in the 2nd portion of the duodenum. Despite 3 times of endoscopic mucosal resections, mucosal local recurrence was found. The depth of the tumour involvement continued to be limited within the mucosal layer. We performed segmental duodenal resection with nodal dissection sacrificing the minor papilla, while preserving the pancreas and the major papilla. The pathological diagnosis was primary intramucosal adenocarcinoma; the surgical margin was negative for cancer and there was no nodal metastasis. This procedure can be an alternative to pancreaticoduodenectomy in patients with earlystage adenocarcinoma in the 2nd portion of the duodenum when the major papilla can be spared, especially in high-risk patients.


Chronic Adherence to a Ketogenic Diet Modifies Iron Metabolism in Elite Athletes.
The short-term restriction of carbohydrate (CHO) can potentially influence iron regulation via modification of postexercise interleukin-6 (IL-6) and hepcidin levels. This study examined the effect of a chronic ketogenic low-CHO high-fat (LCHF) diet on iron status and iron-regulatory markers in elite athletes. International-level race walkers (n = 50) were allocated to one of three dietary interventions: (i) a high-CHO diet (n = 16), (ii) a periodized CHO availability (n = 17), or (iii) an LCHF diet (n = 17) while completing a periodized training program for 3 wk. A 19- to 25-km race walking test protocol was completed at baseline and after adaptation, and changes in serum ferritin, IL-6, and hepcidin concentrations were measured. Results from high-CHO and periodized CHO were combined into one group (CHO; n = 33) for analysis. The decrease in serum ferritin across the intervention period was substantially greater in the CHO group (37%) compared with the LCHF (23%) group (P = 0.021). After dietary intervention, the postexercise increase in IL-6 was greater in LCHF (13.6-fold increase; 95% confidence interval [CI] = 7.1-21.4) than athletes adhering to a CHO-rich diet (7.6-fold increase; 95% CI = 5.5-10.2; P = 0.033). Although no significant differences occurred between diets, CI values indicate that 3 h postexercise hepcidin concentrations were lower after dietary intervention compared with baseline in CHO (β = -4.3; 95% CI = -6.6 to -2.0), with no differences evident in LCHF. Athletes who adhered to a CHO-rich diet experienced favorable changes to the postexercise IL-6 and hepcidin response, relative to the LCHF group. Lower serum ferritin after 3 wk of additional dietary CHO might reflect a larger more adaptive hematological response to training.