Interactions of mucins with the Tn or Sialyl Tn cancer antigens including MUC1 are due to GalNAc-GalNAc interactions.
The molecular mechanism(s) underlying the enhanced self-interactions of mucins possessing the Tn (GalNAcα1-Ser/Thr) or STn (NeuNAcα2-6GalNAcα1-Ser/Thr) cancer markers were investigated using optical tweezers (OT). The mucins examined included modified porcine submaxillary mucin containing the Tn epitope (Tn-PSM), ovine submaxillary mucin with the STn epitope (STn-OSM), and recombinant MUC1 analogs with either the Tn and STn epitope. OT experiments in which the mucins were immobilized onto polystyrene beads revealed identical self-interaction characteristics for all mucins. Identical binding strength and energy landscape characteristics were also observed for synthetic polymers displaying multiple GalNAc decorations. Polystyrene beads without immobilized mucins showed no self-interactions and also no interactions with mucin-decorated polystyrene beads. Taken together, the experimental data suggest that in these molecules, the GalNAc residue mediates interactions independent of the anchoring polymer backbone. Furthermore, GalNAc-GalNAc interactions appear to be responsible for self-interactions of mucins decorated with the STn epitope. Hence, Tn-MUC1 and STn-MUC1 undergo self-interactions mediated by the GalNAc residue in both epitopes, suggesting a possible molecular role in cancer. MUC1 possessing the T (Galβ1-3GalNAcα1-Ser/Thr) or ST antigen (NeuNAcα2-3Galβ1-3GalNAcα1-Ser/Thr) failed to show self-interactions. However, in the case of ST-MUC1, self-interactions were observed after subsequent treatment with neuraminidase and β-galactosidase. This enzymatic treatment is expected to introduce Tn-epitopes and these observations thus further strengthen the conclusion that the observed interactions are mediated by the GalNAc groups.


Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii.
Ruminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1β, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host's immune response.


Weekly irinotecan and concurrent radiation therapy for stage III unresectable NSCLC.
In preclinical studies, the topoisomerase I inhibitor irinotecan (Camptosar, CPT-11) has demonstrated activity as a radiosensitizer, probably due to its ability to inhibit potentially lethal radiation damage repair. We conducted a phase I trial to determine the maximum-tolerated dose (MTD) and dose-limiting toxicities (DLT) of weekly irinotecan with concurrent thoracic radiation therapy for patients with unresectable stage III non-small-cell lung cancer. For this study, 13 patients received three dose escalations (from 30 to 40 to 50 mg/m2/wk). At the first dose level, one patient developed grade 5 esophagitis. Accrual was expanded to seven patients. None of the remaining six patients developed esophagitis. At the second dose level (40 mg/m2/wk), the worst toxicity, which developed in one patient, was grade 2 esophagitis. At the third dose level (50 mg/m2/wk), two of three patients developed grade 4 nausea and vomiting; grade 3 or 4 esophagitis also occurred in two patients. Of the 12 evaluable patients, seven achieved a partial response, for an overall response rate of 58%. In conclusion, nausea, vomiting, and esophagitis appear to be the principal DLTs of concurrent weekly irinotecan and thoracic radiation in the outpatient setting. The MTD of concurrent weekly irinotecan with thoracic radiation therapy appears to be 40 mg/m2 weekly for 6 weeks. To confirm the MTD of this combination, this study is still open to accrual at the second dose level (40 mg/m2) in combination with carboplatin.


Optimized peptide vaccines eliciting extensive CD8 T-cell responses with therapeutic antitumor effects.
A major challenge for developing effective therapeutic vaccines against cancer is overcoming immunologic tolerance to tumor-associated antigens that are expressed on both malignant cells and normal tissues. Herein, we describe a novel vaccination approach, TriVax, that uses synthetic peptides representing CD8 T-cell epitopes, Toll-like receptor agonists that function as potent immunologic adjuvants and costimulatory anti-CD40 antibodies to generate large numbers of high-avidity antigen-reactive T cells capable of recognizing and killing tumor cells. Our results show that TriVax induced huge numbers of long-lasting antigen-specific CD8 T cells that displayed significant antitumor effects in vivo. The administration of a TriVax formulation containing a CD8 T-cell epitope derived from a melanosomal antigen (Trp2(180-188)) elicited antigen-specific CD8 T cells that induced systemic autoimmunity (vitiligo). More important, TriVax immunization was effective in eliciting potent protective antitumor immunity as well as remarkable therapeutic effects against established B16 melanoma. This therapeutic effect was mediated by CD8 T cells via perforin-mediated lysis and required the participation of type-I IFN but not IFNgamma. These results suggest that similar strategies would be applicable for the design of effective vaccination for conducting clinical studies in cancer patients.


Phosphoproteomic analyses reveal novel cross-modulation mechanisms between two signaling pathways in yeast.
Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen-activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time-resolved phosphorylation site dynamics after pathway co-stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone-induced down-regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways.


Protective capacity and epitope specificity of CD8(+) T cells responding to lethal West Nile virus infection.
West Nile virus (WNV) is a small, positive-strand RNA virus belonging to the Flaviviridae genus, which causes lethal encephalitis in a subset of infected birds and mammals. In humans, WNV exhibits pronounced age-related morbidity and mortality, but the basis of this effect is unclear, and the molecular and cellular parameters of the host-WNV infection are just beginning to be elucidated. Indeed, numerous mechanisms were implicated in protection in vivo against WNV (IFN-I and IFN-gamma, antibody, C', CD8 and CD4 T cells), but the individual importance of each one of these remains unclear. Here, we show that transfer of highly enriched naïve CD8(+ )T cells protects the majority of alymphoid mice against lethal WNV infection. To substantiate and expand this finding, we defined the peptide specificity of the CD8 response in H-2b mice and used a panel of identified peptides to map one dominant (NS4b (2248-2256)) and several subdominant epitopes. The hierarchy of these epitopes was stably maintained in the memory responses. Most importantly, CTL lines directed against these peptides conferred protection against lethal WNV infection in direct proportion to the epitope immunodominance. These results provide a springboard for future characterization of T cell responses against WNV and demonstrate, for the first time, that CD8 T cells can single-handedly protect from this disease.


Risks and benefits of ventilation tubes and hearing aids from the perspective of parents of children with cleft palate.
To understand parents' experiences of treatment of otitis media with effusion (OME) for children with cleft palate. Qualitative interviews with parents of 37 children who had non-syndromic cleft palate (with or without cleft lip). They were recruited from two cleft centres in northern England. Participants talked about choices, information, decision-making and satisfaction with ventilation tubes (VTs) and/or hearing aids (HAs). Interviews were recorded and transcribed verbatim. Themes were developed using Framework Analysis. VTs and HAs were not considered as equal treatments by interviewees, due to physical risks associated with the former and social consequences with the latter. The inequality was explained within three main themes: (1) treatment recommendations - most parents did not recall being offered options when OME was first treated; VTs tended to be presented initially followed by HAs if VTs had been inserted more than once. Treatment recommendations came from medical specialists, although participants could also be influenced by other parents' stories; (2) beliefs about mechanisms of treatments - interviewees believed VTs could address the underlying cause of OME by draining fluid. Some parents felt HAs signified a deterioration in hearing and an escalation of care. Improvements in hearing were reported by interviewees with VTs and HAs; (3) demands on parents - participants often saw the insertion of VTs as opportunistic, alongside another procedure (e.g. palatal closure). They could feel disappointed when VTs fell out or were perceived to have caused ear infections. Parental involvement was on-going for HAs (e.g. ordering batteries and tubes). Parents expressed fears about the potential for social stigma of HAs, although none reported significant teasing of children because they wore HAs. Parents' views about treatment for OME were shaped by differing perceptions about anticipated risks and benefits. VTs may have been seen as a simple fix, inserted alongside another procedure, but some interviewees had concerns about possible physical consequences. Others were more worried about anticipated social stigma associated with HAs. However, parents of children who wore a HA described them as easy to manage and well tolerated, at least until children got older and started to comment on their appearance.


ADOR2A C Allele Carriers Exhibit Ergogenic Responses to Caffeine Supplementation.
Caffeine's ergogenic effects on exercise performance are generally explained by its ability to bind to adenosine receptors. ADORA2A is the gene that encodes A2A subtypes of adenosine receptors. It has been suggested that ADORA2A gene polymorphisms may be responsible for the inter-individual variations in the effects of caffeine on exercise performance. In the only study that explored the influence of variation in ADORA2A-in this case, a common polymorphism (rs5751876)-on the ergogenic effects of caffeine on exercise performance, C allele carriers were identified as "non-responders" to caffeine. To explore if C allele carriers are true "non-responders" to the ergogenic effects of caffeine, in this randomized, double-blind study, we examined the acute effects of caffeine ingestion among a sample consisting exclusively of ADORA2A C allele carriers. Twenty resistance-trained men identified as ADORA2A C allele carriers (CC/CT genotype) were tested on two occasions, following the ingestion of caffeine (3 mg/kg) and a placebo. Exercise performance was evaluated with movement velocity, power output, and muscle endurance during the bench press exercise, countermovement jump height, and power output during a Wingate test. Out of the 25 analyzed variables, caffeine was ergogenic in 21 (effect size range: 0.14 to 0.96). In conclusion, ADORA2A (rs5751876) C allele carriers exhibited ergogenic responses to caffeine ingestion, with the magnitude of improvements similar to what was previously reported in the literature among samples that were not genotype-specific. Therefore, individuals with the CT/CC genotype may still consider supplementing with caffeine for acute improvements in performance.


Effects of daily iron supplementation in primary-school-aged children: systematic review and meta-analysis of randomized controlled trials.
Anemia is an important public health and clinical problem. Observational studies have linked iron deficiency and anemia in children with many poor outcomes, including impaired cognitive development; however, iron supplementation, a widely used preventive and therapeutic strategy, is associated with adverse effects. Primary-school-aged children are at a critical stage in intellectual development, and optimization of their cognitive performance could have long-lasting individual and population benefits. In this study, we summarize the evidence for the benefits and safety of daily iron supplementation in primary-school-aged children. We searched electronic databases (including MEDLINE and Embase) and other sources (July 2013) for randomized and quasi-randomized controlled trials involving daily iron supplementation in children aged 5-12 years. We combined the data using random effects meta-analysis. We identified 16 501 studies; of these, we evaluated 76 full-text papers and included 32 studies including 7089 children. Of the included studies, 31 were conducted in low- or middle-income settings. Iron supplementation improved global cognitive scores (standardized mean difference 0.50, 95% confidence interval [CI] 0.11 to 0.90, p = 0.01), intelligence quotient among anemic children (mean difference 4.55, 95% CI 0.16 to 8.94, p = 0.04) and measures of attention and concentration. Iron supplementation also improved age-adjusted height among all children and age-adjusted weight among anemic children. Iron supplementation reduced the risk of anemia by 50% and the risk of iron deficiency by 79%. Adherence in the trial settings was generally high. Safety data were limited. Our analysis suggests that iron supplementation safely improves hematologic and nonhematologic outcomes among primary-school-aged children in low- or middle-income settings and is well-tolerated.


Transfer and isomerization of the ribose moiety of AdoMet during the biosynthesis of queuosine tRNAs, a new unique reaction catalyzed by the QueA protein from Escherichia coli.
The enzyme QueA of E coli is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. The enzyme catalyzes the synthesis of an epoxycyclopentane moiety and transfers this compound to specific tRNAs containing the queuosine precursor 7-(aminomethyl)-7-deazaguanine (preQ1). S-adenosylmethionine (AdoMet) is the sole cofactor that is required for this reaction (Slany et al, 1993, Biochemistry 32, 7811-7817). To proof that the ribose moiety of AdoMet is the precursor of the epoxycyclopentane moiety, labeled AdoMet, was generated from different types of 3H ATP and methionine by the AdoMet synthetase enzyme (MetK) from E coli. The resulting 3H labeled AdoMet was directly used as the cofactor for the QueA reaction. Using [2,5', 8-3H]ATP, containing tritium at C5' of the ribose ring, resulted in an incorporation of radioactivity into preQ1 tRNA, whereas this was not the case when [2,8-3H]ATP was applied. A model for the reaction catalyzed by the S-adenosylmethionine:tRNA ribosyltransferase-isomerase QueA is proposed.


A complex nucleoprotein structure involved in activation of transcription of two divergent Escherichia coli promoters.
Initiation of transcription at malEp and malKp, two divergent Escherichia coli promoters, depends on the presence of both CRP, a pleiotropic activator, and MalT, the maltose regulon activator. We carried out in vivo genetic and functional analysis of these promoters and characterized their interaction with MalT and CRP using DNase I footprinting. The functional limits of the promoters are located about 240 base-pairs (bp) upstream of their transcription start sites, which are 271 bp apart. These promoters therefore overlap by about 210 bp. The overlapping region encompasses four CRP-binding sites and at least four MalT-binding sites. Insertions in the centre of this region are tolerated provided that they correspond to an integral number of DNA helix turns. In DNase I footprinting experiments performed on the complex formed by MalT with malEp-malKp, the DNA appears to be wrapped around the protein. We propose a model for the nucleoprotein structure that might be involved in transcription activation at these divergent promoters.


Immunohistochemical localization of placental leucine aminopeptidase/oxytocinase in normal human placental, fetal and adult tissues.
While oxytocinase is known to exist in pregnancy serum and placenta, the present study describes the expression of the mRNA for this enzyme in a wide variety of other human tissues. Northern blot analysis was used to detect the mRNA, with a probe derived from a cDNA for oxytocinase/placental leucine aminopeptidase (P-LAP). Both the distribution and localization of immunoreactive oxytocinase/P-LAP protein have been determined immunohistochemically by use of an anti-P-LAP antibody in normal placental, fetal and adult tissues. In placental tissues, only syncytiotrophoblasts were stained positively. In both fetal and adult tissues, positive staining was obtained in vascular endothelial cells, gastrointestinal mucosal cells, epithelial cells of hepato-biliary, pancreato-biliary, bronchial-alveolar and renal tubular systems as well as islet cells of pancreas and neurons in the central nervous systems. Sweat-gland cells, seminal vesicles and prostate gland in the adult, as well as adipocytes and skeletal muscle cells in the fetus were also stained. The widespread distribution of P-LAP suggests its involvement in a variety of physiological events not restricted to the regulation of the amounts of bioactive peptides such as arginine vasopressin (AVP) and oxytocin (OT) in pregnancy. The presence of P-LAP in syncytiotrophoblasts supports the idea that P-LAP in pregnancy serum is derived from the placenta.


Soot volume fraction and particle size measurements with laser-induced incandescence.
Laser-induced incandescence from soot was analyzed with a time-dependent, numerical model of particle heating and cooling processes that includes spatial and temporal intensity profiles associated with laser sheet illumination. For volume fraction measurements, substantial errors result primarily from changes in gas temperature and primary soot particle size. The errors can be reduced with the proper choice of detection wavelength, prompt gating, and high laser intensities. Two techniques for primary particle size measurements, based on ratios of laser-induced incandescence signals from a single laser pulse, were also examined. Compared with the ratio of two integration times, the newly proposed ratio of two detection wavelengths is better suited for simultaneous volume fraction and size measurements, because it is less temperature sensitive and produces stronger signals with, however, a lower sensitivity to size changes.


Quantified postsurgical small cell size CTCs and EpCAM+ circulating tumor stem cells with cytogenetic abnormalities in hepatocellular carcinoma patients determine cancer relapse.
Detection of hepatocellular carcinoma circulating tumor cells performed with conventional strategies, is significantly limited due to inherently heterogeneous and dynamic expression of EpCAM, as well as degradation of cytokeratins during epithelial-to-mesenchymal transition, which inevitably lead to non-negligible false negative detection of such "uncapturable and invisible" CTCs. A novel SE-iFISH strategy, improved for detection of HCC CTCs in this study, was applied to comprehensively detect, in situ phenotypically and karyotypically characterize hepatocellular and cholangiocarcinoma CTCs (CD45-/CD31-) in patients subjected to surgical resection. Clinical significance of diverse subtypes of CTC was systematically investigated. Existence of small cell size CTCs (≤5 μm of WBCs) with cytogenetic abnormality of aneuploid chromosome 8, which constituted majority of the detected CTCs in HCC patients, was demonstrated for the first time. The stemness marker EpCAM+ aneuploid circulating tumor stem cells (CTSCs), and EpCAM- small CTCs with trisomy 8, promote tumor growth. Postsurgical quantity of small triploid CTCs (≥5 cells/6 ml blood), multiploid (≥pentasomy 8) CTSCs or CTM (either one ≥ 1) significantly correlated to HCC patients' poor prognosis, indicating that detection of those specific subtypes of CTCs and CTSCs in post-operative patients help predict neoplasm recurrence.


Evaluating children with fractures for child physical abuse.
Fractures are common injuries caused by child abuse. Although the consequences of failing to diagnose an abusive injury in a child can be grave, incorrectly diagnosing child abuse in a child whose fractures have another etiology can be distressing for a family. The aim of this report is to review recent advances in the understanding of fracture specificity, the mechanism of fractures, and other medical diseases that predispose to fractures in infants and children. This clinical report will aid physicians in developing an evidence-based differential diagnosis and performing the appropriate evaluation when assessing a child with fractures.


Safety and efficacy of intravesical alum for intractable hemorrhagic cystitis: A contemporary evaluation.
Hemorrhagic cystitis (HC) represents a challenging clinical entity. While various intravesical agents have been utilized in this setting, limited data exist regard¬ing safety or efficacy. Herein, then, we evaluated the effectiveness and complications associated with intravesical alum instillation for HC in a contemporary cohort. We identified 40 patients treated with intravesical alum for HC between 1997-2014. All patients had failed previous continuous bladder irrigation with normal saline and clot evacuation. Treatment success was defined as requiring no additional therapy beyond normal saline irrigation after alum instillation. Median patient age was 76.5 years (IQR 69, 83). Pelvic radiation was the most common etiology for HC (n=38, 95%). Alum use decreased patient's transfusion requirement, with 82% (32/39) receiving a transfusion within 30 days before alum instillation (median 4 units) versus 59% (23/39) within 30 days after completing alum (median 3 units) (p=0.05). In total, 24 patients (60%) required no additional therapy prior to hospital discharge. Moreover, at a median follow-up of 17 months (IQR 5, 38.5), 13 patients (32.5%) remained without additional treatment for HC. Adverse ef¬fects were reported in 15 patients (38%), with bladder spasms representing the most common event (14/40; 35%). No clinical evidence of clinically significant systemic absorption was detected. Intravesical alum therapy is well-tolerated, with resolution of HC in ap¬proximately 60% of patients, and a durable response in approximately one-third. Given its favorable safety/efficacy profile, intravesical alum may be considered as a first-line treatment option for patients with HC.


Purkinje cell survival is differentially regulated by metabotropic and ionotropic excitatory amino acid receptors.
We previously reported that trophic factors and neurotransmitters in concert regulate survival of cultured cerebellar Purkinje cells. In particular, excitatory amino acid (EAA) transmitters and NGF increased survival, whereas neither alone was effective. In the present studies, we sought to identify molecular mechanisms through which EAAs participate in the survival-promoting interaction. Initially, we characterized the potential role of ionotropic EAA receptors by exposing cultures to the antagonists MK-801, D-2-amino-5-phosphonovaleric acid, and 6,7-dinitroquinoxalinedione. Each increased cell number, suggesting that endogenous ionotropic activity decreased survival. To determine whether metabotropic EAA receptor stimulation modulates survival, the metabotropic agonist ACPD ([1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid; 1 microM) was tested. ACPD alone had no effect on survival. However, simultaneous exposure to ACPD and NGF significantly increased Purkinje number. Moreover, this increase in survival was blocked by L-AP3 [L(+)-2-amino-3-phosphonopropionic acid; 1 microM], a putative antagonist of certain metabotropic responses. L-AP3 also reduced cell number in the absence of exogenous EAA. Thus, endogenous metabotropic stimulation is normally necessary for survival. In sum, these studies reveal a novel mechanism whereby an excitatory neurotransmitter shapes neural development by simultaneous trophic and regressive actions that are, respectively, mediated by metabotropic and ionotropic EAA receptors.


Tobacco use and cessation counselling: Global Health Professionals Survey Pilot Study, 10 countries, 2005.
One of the strategies to reduce the number of smoking-related deaths is to encourage the involvement of health professionals in tobacco-use prevention and cessation counselling. The World Health Organization, the US Centers for Disease Control and Prevention, and the Canadian Public Health Association developed the Global Health Professionals Survey (GHPS) to collect data on tobacco use and cessation counselling among health-profession students in all WHO member states. This report summarises findings from the GHPS Pilot Study, which consisted of 16 surveys conducted in 10 countries among third year students in four health-profession disciplines (dentistry, medicine, nursing, and pharmacy) during the first quarter of 2005. The findings indicated that current cigarette smoking among these students was higher than 20% in seven of the 10 countries surveyed. Nevertheless, 87-99% of the students surveyed believed they should have a role in counselling patients to quit smoking; only 5-37% of these third-year students had actually received formal training in how to conduct such counselling. Schools for health professionals, public health organisations, and education officials should work together to design and implement training in smoking cessation counselling for all health-profession students.


Protocadherin Pcdh2 shows properties similar to, but distinct from, those of classical cadherins.
Cell adhesion and several other properties of a recently identified cadherin-related protein, protocadherin Pcdh2, were characterized. A chimeric Pcdh2 in which the original cytoplasmic domain was replaced with the cytoplasmic domain of E-cadherin was expressed in mouse L cells. The expressed protein had a molecular mass of about 150 kDa and was localized predominantly at the cell periphery, as was the wild-type Pcdh2. In a conventional cell aggregation assay, the transfectants showed cell aggregation activity comparable to that of classical cadherins. This activity was Ca(2+)-dependent and was inhibited by the addition of anti-Pcdh2 antibody, indicating that the chimeric Pcdh2, and probably the wild-type Pcdh2, has Ca(2+)-dependent cell aggregation activity. Mixed cell aggregation assay using L cells and different types of transfectants showed that the activity of Pcdh2 was homophilic and molecular type specific and that Pcdh2 was transfectants did not aggregate with other types of transfectants or with L cells. In immunoprecipitation, the chimeric Pcdh2 co-precipitated with a 105 kDa and a 95 kDa protein, whereas wild-type Pcdh2 co-precipitated with no major protein. Pcdh2 was easily solubilized with non-ionic detergent, in contrast to the case of classical cadherins. On immunofluorescence microscopy, the somas of Purkinje cells were diffusely stained with anti-human Pcdh2 antibody. Mouse Pcdh1 and Pcdh2 were mapped to a small segment of chromosome 18, suggesting that various protocadherins form a gene cluster at this region. The present results suggest that Pcdh2, and possibly other protocadherins as well as protocadherin-related proteins such as Drosophila fat, mediate Ca(2+)-dependent and specific homophilic cell-cell interaction in vivo and play an important role in cell adhesion, cell recognition, and/or some other basic cell processes.


Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors.
Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such "Cys-less" receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the "Cys-loop" proline is absolutely conserved, suggesting the more fitting name "Pro loop" for that motif, and "Pro-loop receptors" for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes.


The minimum element of a synthetic peptide required to block prostate tumor cell migration.
Human prostate tumor cell invasion and metastasis are dependent in part on cell adhesion to extracellular matrix proteins and cell migration. We previously identified a synthetic D-amino acid tumor cell adhesion peptide called HYD1 (kikmviswkg) that supported adhesion of tumor cells derived from breast, prostate, ovary and pancreas tissue. Alanine substitution analysis and a peptide deletion strategy were used to determine the minimal element of HYD1 necessary for bioactivity in a prostate cancer cell line called PC3N. Bioactivity was measured by assays of cell adhesion, migration and ERK signaling. The most potent element of HYD1 necessary to support cell adhesion was kmvixw, the block to migration required xkmviswxx and activation of ERK signaling required ikmviswxx. The shortest sequence active in all three assays was iswkg. The HYD1 peptide contains overlapping elements required for adhesion, blocking migration and the activation of ERK signaling. These linear peptide sequences provide the starting point for development of novel compounds to target cancer cell adhesion and migration.


Protein structure prediction: selecting salient features from large candidate pools.
We introduce a parallel approach, "DT-SELECT," for selecting features used by inductive learning algorithms to predict protein secondary structure. DT-SELECT is able to rapidly choose small, nonredundant feature sets from pools containing hundreds of thousands of potentially useful features. It does this by building a decision tree, using features from the pool, that classifies a set of training examples. The features included in the tree provide a compact description of the training data and are thus suitable for use as inputs to other inductive learning algorithms. Empirical experiments in the protein secondary-structure task, in which sets of complex features chosen by DT-SELECT are used to augment a standard artificial neural network representation, yield surprisingly little performance gain, even though features are selected from very large feature pools. We discuss some possible reasons for this result.


HLA-DP control of human Schistosoma haematobium infection.
The DPA1 and DPB1 alleles of the major histocompatibility complex (MHC) class II were determined in 110 patients and 120 healthy controls of a Gabonese population from an area endemic for Schistosoma haematobium infection. The MHC-DP alleles of the variable second exons and their human leukocyte antigen (HLA) epitopes were correlated with egg excretion, interleukin-4 and interferon-gamma patterns, and bladder abnormalities, as detected by ultrasonography. A methionine at position 11 of the DP alpha molecule (Met-11) and DPA1*0301 were associated with schistosomiasis when compared with controls (phenotypic gene frequencies = 0.791 versus 0.583 and 0.555 versus 0.375, respectively). Met-11 homozygosity occurred more often in patients, whereas healthy controls were more frequently homozygous for an alanine at position 11 (Ala-11). The combination of the DPB1-epitope DEAV (positions 84-87 of the DP beta molecule) and Met-11 positive DPA1 alleles was more frequent in patients than in controls (0.573 versus 0.316). Two years after antischistosomal treatment, the rate of reinfection as examined in 55 of the 110 former patients was higher in DPA1*0301-positive individuals than in those not possessing this allele (P < 0.001). Ala-11 positive individuals showed less frequently ultrasonographic signs of bladder pathology than Ala-11 negative individuals (P < 0.05). Our results suggest a role of MHC-DP elements in the manifestation of disease in S. haematobium infection.


A System-Level Approach to Overweight and Obesity in the Veterans Health Administration.
Healthcare systems are challenged by steady increases in the number of patients who are overweight and obese. Large-scale, evidence-based behavioral approaches for addressing overweight and obesity have been successfully implemented in systems such as the Veterans Health Administration (VHA). These population-based interventions target reduction in risk for obesity-associated conditions through lifestyle change and weight loss, and are associated with modest weight loss. Despite the fact that VHA has increased the overall reach of these behavioral interventions, the number of high-risk overweight and obese patients continues to rise. Recommendations for weight loss medications and bariatric surgery are included in clinical practice guidelines for the management of overweight and obesity, but these interventions are underutilized. During a recent state of the art conference on weight management held by VHA, subject matter experts identified challenges and gaps, as well as potential solutions and overarching policy recommendations, for implementing an integrated system-wide approach for improving population-based weight management.


Bacterial community structure associated with white band disease in the elkhorn coral Acropora palmata determined using culture-independent 16S rRNA techniques.
Culture-independent molecular (16S ribosomal RNA) techniques showed distinct differences in bacterial communities associated with white band disease (WBD) Type I and healthy elkhorn coral Acropora palmata. Differences were apparent at all levels, with a greater diversity present in tissues of diseased colonies. The bacterial community associated with remote, non-diseased coral was distinct from the apparently healthy tissues of infected corals several cm from the disease lesion. This demonstrates a whole-organism effect from what appears to be a localised disease lesion, an effect that has also been recently demonstrated in white plague-like disease in star coral Montastraea annularis. The pattern of bacterial community structure changes was similar to that recently demonstrated for white plague-like disease and black band disease. Some of the changes are likely to be explained by the colonisation of dead and degrading tissues by a micro-heterotroph community adapted to the decomposition of coral tissues. However, specific ribosomal types that are absent from healthy tissues appear consistently in all samples of each of the diseases. These ribotypes are closely related members of a group of alpha-proteobacteria that cause disease, notably juvenile oyster disease, in other marine organisms. It is clearly important that members of this group are isolated for challenge experiments to determine their role in the diseases.


Decentralised facility-based training as an alternative model for SLMTA implementation: The Cameroon experience.
The Strengthening Laboratory Management Toward Accreditation (SLMTA) programme is designed to build institutional capacity to help strengthen the tiered laboratory system. Most countries implement the SLMTA three-workshop series using a centralised model, whereby participants from several laboratories travel to one location to be trained together. We assessed the effectiveness and cost of conducting SLMTA training in a decentralised manner as compared to centralised training. SLMTA was implemented in five pilot laboratories in Cameroon between October 2010 and October 2012 by means of a series of workshops, laboratory improvement projects and on-site mentorship. The first workshop was conducted in the traditional centralised approach. The second and third workshops were decentralised, delivered on-site at each of the five enrolled laboratories. Progress was monitored by repeated audits using the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) checklist. Audit scores for all laboratories improved steadily through the course of the programme. Median improvement was 11 percentage points after the first (centralised) training and an additional 24 percentage points after the second (decentralised) training. The estimated per-laboratory cost of the two training models was approximately the same at US$21 000. However, in the decentralised model approximately five times as many staff members were trained, although it also required five times the amount of trainer time. Decentralised SLMTA training was effective in improving laboratory quality and should be considered as an alternative to centralised training.


Identification of sites influencing the folding and subunit assembly of the P22 tailspike polypeptide chain using nonsense mutations.
Amber mutations have been isolated and mapped to more than 60 sites in gene 9 of P22 encoding the thermostable phage tailspike protein. Gene 9 is the locus of over 30 sites of temperature sensitive folding (tsf) mutations, which affect intermediates in the chain folding and subunit association pathway. The phenotypes of the amber missense proteins produced on tRNA suppressor hosts inserting serine, glutamine, tryosine and leucine have been determined at different temperatures. Thirty-three of the sites are tolerant, producing functional proteins with any of the four amino acids inserted at the sites, independent of temperature. Tolerant sites are concentrated at the N-terminal end of the protein indicating that this region is not critical for conformation or function. Sixteen of the sites yield temperature sensitive missense proteins on at least one nonsense suppressing host. Most of the sites with ts phenotypes map to the central region of the gene which is also the region where most of the tsf mutations map. Mutations at 15 of the sites have a lethal phenotype on at least one tRNA suppressor host. For nine out of ten sites tested with at least one lethal phenotype, the primary defect was in the folding or subunit association of the missense polypeptide chain. This analysis of the tailspike missense proteins distinguishes three classes of amino acid sites in the polypeptide chain; residues whose side chains contribute little to folding, subunit assembly or function; residues critical for maintaining the folding and subunit assembly pathway at the high end of the temperature range of phage growth; and residues critical over the entire temperature range of growth.


An ethnographic study of a day care center for Iranian immigrant seniors.
Late-in-life immigrants are often at risk for psychological stress and social isolation because of language problems, small social networks, and cultural differences from the host society. Community intervention programs can reduce such stress and isolation. In this article, the authors describe a Swedish municipality's culturally appropriate intervention program for elderly Iranian immigrants based on ethnographic data gathered during a 12 month period. The description includes the activities provided by the program and the experiences of the elderly Iranian immigrants who participate regularly in these activities. The findings document the positive impact of regular participation in the center's activities on the elders' well-being and health.


Beginning readers activate semantics from sub-word orthography.
Two experiments assessed whether 7-year-old children activate semantic information from sub-word orthography. Children made category decisions to visually-presented words, some of which contained an embedded word (e.g., hip in ship). In Experiment 1 children were slower and less accurate to classify words if they contained an embedded word related in meaning to the category (e.g., slower to reject ship as a 'body part' than an 'animal'), especially when the embedded word was higher in frequency than the carrier word. This demonstrates that young children activate semantic information from sub-word orthographic representations, and that they do so from the relatively early stages of learning to read. Experiment 2 replicated this effect. Furthermore, we observed semantic interference regardless of whether the embedded word shared its pronunciation with the carrier (e.g., the hip in ship) or not (e.g., the crow in crown), and regardless of its position within the carrier, suggesting that interference was not dependent on phonological mediation. These findings show that by 7-years-of age, children have begun to establish an orthographic system that is capable of activating sub-word orthographic patterns, strong enough to connect with meaning, when reading words silently.


Inherited human cPLA(2alpha) deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction.
Cytosolic phospholipase A2alpha (cPLA2alpha) hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the synthesis of eicosanoids, such as prostaglandins and leukotrienes. Considerable understanding of cPLA2alpha function has been derived from investigations of the enzyme and from cPLA2alpha-null mice, but knowledge of discrete roles for this enzyme in humans is limited. We investigated a patient hypothesized to have an inherited prostanoid biosynthesis deficiency due to his multiple, complicated small intestinal ulcers despite no use of cyclooxygenase inhibitors. Levels of thromboxane B2 and 12-hydroxyeicosatetraenoic acid produced by platelets and leukotriene B4 released from calcium ionophore-activated blood were markedly reduced, indicating defective enzymatic release of the arachidonic acid substrate for the corresponding cyclooxygenase and lipoxygenases. Platelet aggregation and degranulation induced by adenosine diphosphate or collagen were diminished but were normal in response to arachidonic acid. Two heterozygous single base pair mutations and a known SNP were found in the coding regions of the patient's cPLA2alpha genes (p.[Ser111Pro]+[Arg485His; Lys651Arg]). The total PLA2 activity in sonicated platelets was diminished, and the urinary metabolites of prostacyclin, prostaglandin E2, prostaglandin D2, and thromboxane A2 were also reduced. These findings characterize what we believe is a novel inherited deficiency of cPLA2.


Poorer mental health in UK bisexual women than lesbians: evidence from the UK 2007 Stonewall Women's Health Survey.
Bisexual- and lesbian-identified women have significantly worse mental health than heterosexual women. Less evidence exists about mental health differences between lesbian and bisexual women. Self-completion survey with community-based, opportunistic sampling recruited 937 bisexual-identified and 4769 lesbian-identified women. Associations between sexual identity and mental health indicators were assessed by logistic regression, controlling for age, income, student status and employment. As a group, bisexual women were younger, poorer, and more likely to be trans-identified, minority ethnic identified and to use marijuana, compared with lesbians. Bisexuals were more likely than lesbians to report eating problems (adjusted odds ratio (AOR) = 1.64, P < 0.001), self-harm (AOR = 1.37, P = 0.001), depressed feelings (AOR = 1.26, P = 0.022) and anxiety (AOR = 1.20, P = 0.037). Fewer bisexual women attended lesbian or bisexual social events, were 'out', or had experienced any sexuality-related discrimination, compared with lesbians. More bisexual women reported poor mental health or psychological distress than did lesbians. Bisexual women may be more likely to experience social stress due to the 'double discrimination' of homophobia and biphobia. This stress, experienced mainly as internalized and felt stigma, could result in greater risk for poor mental health compared with lesbians. Addressing both biphobia and homophobia within UK society has important preventative mental health implications.


Medullary cystic kidney disease type 1: mutational analysis in 37 genes based on haplotype sharing.
Medullary cystic kidney disease type 1 (MCKD1) is an autosomal dominant, tubulo-interstitial nephropathy that causes renal salt wasting and end-stage renal failure in the fourth to seventh decade of life. MCKD1 was localized to chromosome 1q21. We demonstrated haplotype sharing and confirmed the telomeric border by a recombination of D1S2624 in a Belgian kindred. Since the causative gene has been elusive, high resolution haplotype analysis was performed in 16 kindreds. Clinical data and blood samples of 257 individuals (including 75 affected individuals) from 26 different kindreds were collected. Within the defined critical region mutational analysis of 37 genes (374 exons) in 23 MCKD1 patients was performed. In addition, for nine kindreds RT-PCR analysis for the sequenced genes was done to screen for mutations activating cryptic splice sites. We found consistency with the haplotype sharing hypothesis in an additional nine kindreds, detecting three different haplotype subsets shared within a region of 1.19 Mb. Mutational analysis of all 37 positional candidate genes revealed sequence variations in 3 different genes, AK000210, CCT3, and SCAMP3, that were segregating in each affected kindred and were not found in 96 healthy individuals, indicating, that a single responsible gene causing MCKD1 remains elusive. This may point to involvement of different genes within the MCKD1 critical region.


Good clinical laboratory practices improved proficiency testing performance at clinical trials centers in Ghana and Burkina Faso.
The recent drive towards accreditation of clinical laboratories in Africa by the World Health Organization-Regional Office for Africa (WHO-AFRO) and the U.S Government is a historic step to strengthen health systems, provide better results for patients and an improved quality of results for clinical trials. Enrollment in approved proficiency testing (PT) programs and maintenance of satisfactory performance is vital in the process of accreditation. Passing proficiency testing surveys has posed a great challenge to many laboratories across sub-Saharan Africa. Our study was aimed at identifying the causes of unsatisfactory PT results in clinical research laboratories conducting or planning to conduct malaria vaccine trials sponsored by the National Institutes of Health (NIH). PT reports for 2009 and 2010 from the College of American Pathologists (CAP) for the laboratories were reviewed as part of the process. Errors accounting for unsatisfactory results were classified into clerical, methodological, technical, problem with PT materials, and random errors. A training program on good clinical laboratory practices (GCLP) was developed for each center to address areas for improvement. The major cause of PT failure in the four centers was methodological. The application of GCLP improved the success rate in the PT surveys from 58% in 2009 to 88% in 2010. It also decreased the error rate on PT by 35%. A previous report from the CAP- PT participating laboratories indicated that the major causes of error were clerical. These types of errors were predominantly made in laboratories in the US, with much more experience in quality control, and varied significantly from what we found. In our centers in sub-Saharan Africa, methodological errors, and not clerical errors, accounted for the vast majority of errors. A process was started for continuous improvement which has decreased methodological errors by 35%, but more improvement is needed.


A computational study of CO oxidation reactions on metal impurities in graphene divacancies.
Based on the density functional theory calculations, the formation geometry, electronic properties, and catalytic activity of metal impurities in divacancy graphene (M-DG, M = Mo, Fe, Co, and Ni) were systematically investigated. It has been found that the reactive gases have different stabilities on M-DG substrates, and these quite stable substrates exhibit high catalytic activity for CO oxidation by comparing the traditional Eley-Rideal (ER) and Langmuir-Hinshelwood (LH), as well as the new termolecular ER (TER) mechanisms. For the Co-DG substrate, the coadsorption of O2 and CO as a starting step is an energetically more favorable process, whereas the dissociation reaction of O2 molecules on Mo-DG substrate has a much smaller energy barrier, and the generation of atomic oxygen is active for CO oxidation. These results indicate that the varied adsorption behaviors of reactive gases on M-DG substrates can determine the catalytic pathways and energy barriers, which give us insight into the surface reactivity of graphene-metal composite catalysis in energy-related devices.


In the heartland of Eurasia: the multilocus genetic landscape of Central Asian populations.
Located in the Eurasian heartland, Central Asia has played a major role in both the early spread of modern humans out of Africa and the more recent settlements of differentiated populations across Eurasia. A detailed knowledge of the peopling in this vast region would therefore greatly improve our understanding of range expansions, colonizations and recurrent migrations, including the impact of the historical expansion of eastern nomadic groups that occurred in Central Asia. However, despite its presumable importance, little is known about the level and the distribution of genetic variation in this region. We genotyped 26 Indo-Iranian- and Turkic-speaking populations, belonging to six different ethnic groups, at 27 autosomal microsatellite loci. The analysis of genetic variation reveals that Central Asian diversity is mainly shaped by linguistic affiliation, with Turkic-speaking populations forming a cluster more closely related to East-Asian populations and Indo-Iranian speakers forming a cluster closer to Western Eurasians. The scattered position of Uzbeks across Turkic- and Indo-Iranian-speaking populations may reflect their origins from the union of different tribes. We propose that the complex genetic landscape of Central Asian populations results from the movements of eastern, Turkic-speaking groups during historical times, into a long-lasting group of settled populations, which may be represented nowadays by Tajiks and Turkmen. Contrary to what is generally thought, our results suggest that the recurrent expansions of eastern nomadic groups did not result in the complete replacement of local populations, but rather into partial admixture.


Health care professional perspectives on discharging hospitalized patients with injection drug use-associated infections.
Patients with injection drug use (IDU)-associated infections traditionally experience prolonged hospitalizations, which often result in negative experiences and bad outcomes. Harm reduction approaches that value patient autonomy and shared decision-making regarding outpatient treatment options may improve outcomes. We sought to identify health care professionals (HCPs) perspectives on the barriers to offering four different options to hospitalized people who use drugs (PWUD): long-term hospitalization, oral antibiotics, long-acting antibiotics at an infusion center, and outpatient parenteral antibiotics. We recruited HCPs (n = 19) from a single tertiary care center in Portland, Maine. We interviewed HCPs involved with discharge decision-making and other HCPs involved in the specialized care of PWUD. Semi-structured interviews elicited lead HCP values, preferences, and concerns about presenting outpatient antimicrobial treatment options to PWUD, while support HCPs provided contextual information. We used the iterative categorization approach to code and thematically analyze transcripts. HCPs were willing to present outpatient treatment options for patients with IDU-associated infections, yet several factors contributed to reluctance. First, insufficient resources, such as transportation, may make these options impractical. However, HCPs may be unaware of existing community resources or viable treatment options. They also may believe the hospital protects patients, and that discharging patients into the community exposes them to structural harms. Some HCPs are concerned that patients with substance use disorder will not make 'good' decisions regarding outpatient antimicrobial options. Finally, there is uncertainty about how responsibility for offering outpatient treatment is shared across changing care teams. HCPs perceive many barriers to offering outpatient care for people with IDU-associated infections, but with appropriate interventions to address their concerns, may be open to considering more options. This study provides important insights and contextual information that can help inform specific harm reduction interventions aimed at improving care of people with IDU-associated infections.


Intermittent preventive treatment of malaria in pregnancy: a new delivery system and its effect on maternal health and pregnancy outcomes in Uganda.
To assess whether traditional birth attendants, drug-shop vendors, community reproductive-health workers, or adolescent peer mobilizers could administer intermittent preventive treatment (IPTp) for malaria with sulfadoxine-pyrimethamine to pregnant women. A non-randomized community trial was implemented in 21 community clusters (intervention) and four clusters where health units provided routine IPTp (control). The primary outcome measures were access and adherence to IPTp, number of malaria episodes, prevalence of anaemia, and birth weight. Numbers of live births, abortions, still births, and maternal and child deaths were secondary endpoints. 1404 (67.5%) of 2081 with the new delivery system received two doses of sulfadoxine-pyrimethamine versus 281 (39.9%) of 704 with health units (P < 0.0001). The prevalence of malaria episodes decreased from 906 (49.5%) of 1830 to 160 (17.6%) of 909 (P < 0.001) with the new delivery system and from 161 (39.1%) of 412 to 13 (13.1%) of 99 (P < 0.001) with health units. Anaemia was significantly less prevalent in both arms. There was a lower proportion of low birth weight 6.0% with the new delivery system versus 8.3% with health units (P < 0.03). Few abortions and stillbirths were recorded in either arm. Fewer children and women who accessed IPTp with health units died than in the intervention group. The new approaches were associated with early access and increased adherence to IPTp. Health units were, however, more effective in reducing parasitaemia and malaria episodes. We recommend further studies to assess programming modalities linking the new approaches and health units.


Adaptive neural control of uncertain MIMO nonlinear systems.
In this paper, adaptive neural control schemes are proposed for two classes of uncertain multi-input/multi-output (MIMO) nonlinear systems in block-triangular forms. The MIMO systems consist of interconnected subsystems, with couplings in the forms of unknown nonlinearities and/or parametric uncertainties in the input matrices, as well as in the system interconnections without any bounding restrictions. Using the block-triangular structure properties, the stability analyses of the closed-loop MIMO systems are shown in a nested iterative manner for all the states. By exploiting the special properties of the affine terms of the two classes of MIMO systems, the developed neural control schemes avoid the controller singularity problem completely without using projection algorithms. Semiglobal uniform ultimate boundedness (SGUUB) of all the signals in the closed-loop of MIMO nonlinear systems is achieved. The outputs of the systems are proven to converge to a small neighborhood of the desired trajectories. The control performance of the closed-loop system is guaranteed by suitably choosing the design parameters. The proposed schemes offer systematic design procedures for the control of the two classes of uncertain MIMO nonlinear systems. Simulation results are presented to show the effectiveness of the approach.


Biochemical defense mechanisms against copper-induced oxidative damage in the blue crab, Callinectes sapidus.
The blue crab (Callinectes sapidus) has a very dynamic copper metabolism associated with the biosynthesis and degradation of its respiratory pigment hemocyanin. In this study we report on the cellular defense mechanisms used by the crab to protect itself from copper toxicity. Short-term copper-exposure studies, conducted by incubating hepatopancreas tissue explants in copper-containing medium, show that copper taken up by the cells during the first 60 min combines with low-molecular-weight copper complex(es), which include Cu(I)-glutathione. Thereafter, copper binds to newly synthesized metallothionein (MT), with a concomitant decrease in Cu(I)-glutathione. Copper does not displace zinc from the endogenous ZnMT pool. Long-term exposure by means of copper-rich diets results in the synthesis of two MT isoforms in the hepatopancreas: CuMT-I and CuMT-II (D. Schlenk and M. Brouwer, 1991, Aquat. Toxicol. 20, 25-34). Transfer of copper from Cu(I)-glutathione to apoMT-I and apoMT-II can be accomplished in vitro. Cu(I) binding by the two isoforms is very different. Cu(I) binds to apoMT-I in a strictly cooperative manner. No partially filled Cu(I)-thiolate clusters appear to be present. In contrast, the Cu(I)-thiolate clusters in MT-II are formed only after more than four Cu(I) ions are bound. Long-term copper exposure leads to increased activity of two antioxidant enzymes: glutathione peroxidase and manganese superoxide dismutase (SOD). No CuZnSOD is found. Activities of catalase and glutathione reductase and the intracellular levels of glutathione are unaffected by copper. The defense mechanisms are not entirely sufficient for preventing copper-induced oxidative damage. Levels of oxidized lipids are significantly higher in copper-exposed crabs, but oxidized protein levels are nearly the same.


Lifecourse social position and D-dimer; findings from the 1958 British birth cohort.
The aim is to examine the association of lifecourse socioeconomic position (SEP) on circulating levels of D-dimer. Data from the 1958 British birth cohort were used, social class was determined at three stages of respondents' life: at birth, at 23 and at 42 years. A cumulative indicator score of SEP (CIS) was calculated ranging from 0 (always in the highest social class) to 9 (always in the lowest social class). In men and women, associations were observed between CIS and D-dimer (P<0.05). Thus, the respondents in more disadvantaged social classes had elevated levels of D-dimer compared to respondents in less disadvantaged social class. In multivariate analyses, the association of disadvantaged social position with D-dimer was largely explained by fibrinogen, C-reactive protein and von Willebrand Factor in women, and additionally by smoking, alcohol consumption and physical activity in men. Socioeconomic circumstances across the lifecourse at various stages also contribute independently to raised levels of D-dimer in middle age in women only. Risk exposure related to SEP accumulates across life and contributes to raised levels of D-dimer. The association of haemostatic markers and social differences in health may be mediated by inflammatory and other markers.


Carotenoid cleavage dioxygenase4 is a negative regulator of β-carotene content in Arabidopsis seeds.
Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID cleavage dioxygenase4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.


Effect of Thoracic Epidural Ropivacaine versus Bupivacaine on Lower Urinary Tract Function: A Randomized Clinical Trial.
Thoracic epidural analgesia with bupivacaine resulted in clinically relevant postvoid residuals due to detrusor underactivity. This study aimed to compare the risk of bladder dysfunction with ropivacaine versus bupivacaine using postvoid residuals and maximum flow rates. Our hypothesis was that ropivacaine would result in lower postvoid residuals, because ropivacaine has been shown to have less effect on motor blockade. In this single-center, parallel-group, randomized, double-blind superiority trial, 42 patients undergoing open renal surgery were equally allocated to receive epidural bupivacaine 0.125% or ropivacaine 0.2%, and 36 were finally included. Inclusion criterion was normal bladder function. Patients underwent urodynamic investigations preoperatively and during thoracic epidural analgesia. Primary outcome was the difference in postvoid residual preoperatively and during thoracic epidural analgesia postoperatively. Secondary outcomes were changes in maximum flow rate between and within the groups. Median difference in postvoid residual (ml) from baseline to postoperatively was 300 (range, 30 to 510; P < 0.001) for bupivacaine and 125 (range, -30 to 350; P = 0.011) for ropivacaine, with a significant mean difference between groups (-175; 95% CI, -295 to -40; P = 0.012). Median difference in maximum flow rate (ml/s) was more pronounced with bupivacaine (-12; range, -28 to 3; P < 0.001) than with ropivacaine (-4; range, -16 to 7; P = 0.025) with a significant mean difference between groups (7; 95% CI, 0 to 12; P = 0.028). Pain scores were similar. No adverse events occurred. Postvoid residuals were significantly lower using ropivacaine compared to bupivacaine for thoracic epidural analgesia reflecting less impairment of detrusor function with ropivacaine.


Glycogen synthase kinase-3β regulates anti-inflammatory property of fluoxetine.
A selective serotonin reuptake inhibitor fluoxetine not only is widely used in the treatment of depression but also has an anti-inflammatory property. Glycogen synthase kinase-3beta (GSK-3β) is a vital factor in the inflammation process. How fluoxetine interferes with inflammation via a GSK-3β-dependent pathway remains unclear. The aim of this study is to investigate the effects of fluoxetine on lipopolysaccharide (LPS)-induced inflammation. Results showed that fluoxetine decreased mortality rate of the mice. It also inhibited LPS-induced release of nitric oxide (NO) and prostaglandin E2 (PGE2) in serum and RAW264.7 murine macrophages and expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Upon LPS stimulation, fluoxetine caused a delay but increased in the phosphorylated levels of GSK-3β (ser9), whereas it did not affect LPS-induced activation of mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS). Fluoxetine in combination with phosphatidylinositol 3-kinases/Akt inhibitors (LY294002 and Wortmannin) did not have a synergistic inhibition on LPS-induced NO release and PGE2 production. In addition, peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9622 showed no reverse effects of this inhibition of fluoxetine. GSK-3β knockdown blocked the inhibitory effects of fluoxetine on LPS-induced iNOS/NO release and COX-2/PGE2 production. These results indicated that GSK-3β regulated anti-inflammatory property of fluoxetine. However, Akt activation, ROS generation, and altered PPARγ activity were not involved in this inhibition of fluoxetine.


Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories.
This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).


Skin Intrinsic Fluorescence and Age-Related Macular Degeneration: The Beaver Dam Eye Study.
To determine if skin intrinsic fluorescence (SIF), a noninvasive measure of advanced glycation endproducts and oxidative stress in skin is associated with AMD. SIF was measured with the SCOUT DS skin fluorescence spectrometer in a cross-sectional cohort study of 969 persons aged 68 to 102 years from the 1181 who participated in the 25-year follow-up examination in the Beaver Dam Eye Study (BDES) in 2014 to 2016. The SCOUT DS skin fluorescence spectrometer uses five light-emitting diodes, centered at 375 nm to 456 nm. AMD was assessed by grading of digital color 45° stereoscopic fundus photographs of the macula using the Wisconsin Age-Related Maculopathy grading scheme. Analyses included logistic regression with generalized estimating equations to account for correlation between the eyes of a person. There were data for 1827 eyes for analyses. Early AMD was present in 22% and late AMD in 4% of the eyes. While adjusting for age, sex, smoking status, and history of cardiovascular disease, there were no significant associations of any SIF measure with any AMD or exudative AMD. SIF01 (odds ratio per 1 SD difference on the log scale, 95% confidence interval) (1.66, 1.00-2.74, P = 0.05) and SIF03 (1.81, 1.16-2.81, P = 0.008) were associated with geographic atrophy. There was a suggestive relationship of two SIF measures, SIF01 and SIF03, using different correction factors from the excitation centered at 375 nm, with the prevalence of geographic atrophy in the BDES. Longitudinal follow-up is indicated to assess a temporal relationship.


Brain-Derived Neurotrophic Factor in the Nucleus Accumbens Mediates Individual Differences in Behavioral Responses to a Natural, Social Reward.
BDNF-oxytocin interactions in the brain are implicated in mammalian maternal behavior. We found that BDNF gene expression is increased in the hippocampus of rat mothers that show increased pup licking/grooming (high LG mothers) compared to low LG mothers. High LG mothers also showed increased BDNF protein levels in the nucleus accumbens (nAcc). Immunoneutralization of BDNF in the nAcc eliminated the differences in pup LG between high and low LG mothers. Oxytocin antagonist in the ventral hippocampus significantly decreased the frequency of maternal LG behavior. Oxytocin antagonist significantly prevented the oxytocin-induced BDNF gene expression in primary hippocampal cell cultures. We suggest that oxytocin-induced regulation of BDNF in the nAcc provides a neuroendocrine basis for both individual differences in maternal behavior and resilience to the stress of reproduction in female mammals.


Differences in risk profiles and long-term outcomes in acute heart failure patients with preserved and reduced left ventricular ejection fraction in the Czech Republic: The AHEAD registry sub-analysis.
The latest European heart failure guidelines define patients as those with reduced (HFrEF), mid-range, and preserved (HFpEF) left ventricular ejection fraction (LVEF; <40%, 40%-49%, and ≥50%, respectively). We investigated the causes of rehospitalizations/deaths in our institution's heart failure patients and focused on differences in the clinical presentation, risk profile, and long-term outcomes between the HFrEF and HFpEF groups in a real-life scenario. We followed 1274 patients discharged from heart failure hospitalization in 2 centres. The mean patient age was 75.9 years, and men and women were represented equally. During the minimal follow-up of 2 years, 57% of patients were hospitalised for any cause, 24.9% for decompensated heart failure, and 43.3% for any cardiovascular cause. A total of 36.1% of patients died, either with prior (11.8%) or without prior (24.3%) heart failure rehospitalization. Heart failure was also the most frequent cause of cardiovascular hospitalization, followed by gastrointestinal problems, infections, and tumours for noncardiovascular hospitalizations. Patients with HFrEF had different baseline characteristics and risk profiles, experienced more hospitalizations for acute heart failure (28.6% vs 20.2%, P=0.012), and had higher cardiovascular mortality (82.4% vs 63.5%, P<0.001) when compared with HFpEF patients. Overall mortality and rehospitalization rates were similar. Within 2 years, half of the patients died and/or were hospitalised for acute decompensation of heart failure, and only one-third of the patients survived without any hospitalization. HFrEF and HFpEF patients were confirmed to be different entities with diverse characteristics, risk profiles, and cardiovascular event rates.


Comparison between a newly developed PC-based Doppler umbilical artery waveform analyser and a commercial unit.
To determine the accuracy of the resistance index (RI) of flow velocity waveforms of the umbilical artery measured using a newly developed PC-based continuous wave Doppler device (Umbiflow) with regard to systematic and random variations when compared with a commercial standard (Vasoflow). A cohort study. The fetal evaluation clinic (FEC) at Tygerberg Hospital. Patients referred to the FEC at Tygerberg Hospital with suspected chronic placental insufficiency. The correlation coefficients indicating the strength of the relationship between the two devices and their agreement using the method of Bland and Altman. A total of 248 patients were studied. The mean RIs of the first Doppler assessment were 0.69 (standard deviation (SD) 0.11) and 0.67 (SD 0.11) using the Vasoflow and Umbiflow respectively. The Pearson's correlation coefficient comparing the RI of the first test was 0.85. The degree of agreement between the two methods was excellent, the mean differences being very small (< 0.024) with tight confidence intervals. One hundred and ninety-four patients (78.2%) of patients remained in the same percentile category with both the Vasoflow and Umbiflow. The accuracy of the Umbiflow has been proved. A non-significant trend towards slightly lower values needs to be considered. This could be addressed by changing the percentile cut-off to slightly lower values.


Health status of returning refugees, internally displaced persons, and the host community in a post-conflict district in northern Sri Lanka: a cross-sectional survey.
Although the adverse impacts of conflict-driven displacement on health are well-documented, less is known about how health status and associated risk factors differ according to displacement experience. This study quantifies health status and quality of life among returning refugees, internally displaced persons, and the host community in a post-conflict district in Northern Sri Lanka, and explores associated risk factors. We analysed data collected through a household survey (n = 570) in Vavuniya district, Sri Lanka. The effect of displacement status and other risk factors on perceived quality of life as estimated from the 36-item Short Form Questionnaire, mental health status from 9-item Patient Health Questionnaire, and self-reported chronic disease status were examined using univariable analyses and multivariable regressions. We found strong evidence that perceived quality of life was significantly lower for internally displaced persons than for the host community and returning refugees, after adjusting for covariates. Both mental health status and chronic disease status did not vary remarkably among the groups, suggesting that other risk factors might be more important determinants of these outcomes. Our study provides important insights into the overall health and well-being of the different displaced sub-populations in a post-conflict setting. Findings reinforce existing evidence on the relationship between displacement and health but also highlight gaps in research on the long-term health effects of prolonged displacement. Understanding the heterogeneity of conflict-affected populations has important implications for effective and equitable humanitarian service delivery in a post-conflict setting.


Prevention and treatment produced large decreases in HIV incidence in a model of people who inject drugs.
In the United States, people who inject drugs continue to be at greatly increased risk of HIV infection. To estimate the effectiveness of various prevention scenarios, we modeled HIV transmission in a dynamic network of drug users and people who did not use drugs that was based on the New York Metropolitan Statistical Area population. We compared the projected HIV incidence in 2020 and 2040 if current approaches continue to be used to the incidence if one or more of the following hypothetical interventions were applied: increased HIV testing, improved access to substance abuse treatment, increased use of needle and syringe programs, scaled-up treatment as prevention, and a "high impact" combination scenario, consisting of all of the strategies listed above. No strategy completely eliminated HIV transmission. The high-impact combination strategy produced the largest decrease in HIV incidence-a 62 percent reduction compared to the status quo. Our results suggest that increased resources for and investments in multiple HIV prevention approaches will be required to eliminate HIV transmission among people who inject drugs.


Effectiveness and tolerability of melatonin and zolpidem for the alleviation of jet lag.
The aim of this study was to compare the effectiveness and tolerability of a chronobiotic (melatonin) with a hypnotic (zolpidem) and the combination of both substances to alleviate jet lag symptoms associated with eastward travel. This double-blind, randomized, placebo-controlled study is based on 137 volunteers flying from Switzerland to the American continent and back (6-9 time zones). The participants either received melatonin 5 mg (n = 35), zolpidem 10 mg (n = 34), a combination thereof (n = 29) or placebo (n = 39) on the eastbound flight back to Switzerland and once daily at bedtime on 4 consecutive days after the flight. The test battery included daily sleep logs, symptoms questionnaires, and the Profile of Mood States (POMS). Also, on the last treatment day, Visual Analog Scales (VAS) were completed to assess overall jet lag ratings and treatment effectiveness. Baseline data were collected on 4 consecutive days 2 wk after the flight. During post-flight treatment and baseline, motor activity was assessed in a subgroup of 49 subjects using wrist-worn ambulatory monitors. The self-rated sleep quality was significantly improved by zolpidem, especially during the night flight. Subjects taking zolpidem reported significantly less jet lag and zolpidem was rated as the most effective jet lag medication. However, zolpidem and the combination melatonin/zolpidem were less well tolerated than melatonin alone; adverse event reports included nausea, vomiting, amnesia and somnambulia to the point of incapacitation. Confusion, morning sleepiness and nausea were highest in the combination group. All active treatments led to a decrease of jet lag severity with zolpidem being the most effective treatment, particularly in facilitating sleep on night flights. Potential individual adverse reactions to this hypnotic have to be considered.


Association Between Glycemic Control and Risk of Fracture in Diabetic Patients: A Nested Case-Control Study.
Diabetes mellitus (DM) has been associated with an increased risk of fractures. However, the effect of glycemic control on the risk of fracture is not well understood. To evaluate the association between glycemic control and the risk of low-trauma fractures in patients with type 1 DM (T1DM) and type 2 DM (T2DM). Nested case-control analysis. UK-based Clinical Practice Research Datalink. The study population was patients whose T1DM or T2DM had been newly diagnosed between 1995 and 2015. The cases were patients with a low-trauma fracture after DM onset. We matched four controls to each case by age, sex, general practice, fracture date, and DM type and duration. Conditional logistic regression analyses were performed, adjusted for covariates, including body mass index, smoking, DM complications and medications. The study population included 3329 patients with T1DM and 44,275 patients with T2DM. The median duration between DM onset and fracture date was 4.5 years for both T1DM and T2DM. The risk of fracture was increased in the patients with T1DM with a mean hemoglobin A1c >8.0% (adjusted OR, 1.39; 95% CI, 1.06 to 1.83) compared with those patients with T1DM and a mean hemoglobin A1c ≤7.0%. No such effect was found in the patients with T2DM. Independently of glycemic control, the risk of fracture was elevated in patients with T2DM and the current use of rosiglitazone and pioglitazone. The effect of glycemic control on the risk of low-trauma fracture differs between patients with T1DM and T2DM. Poor glycemic control increased the risk of fractures in patients with T1DM but not in those with T2DM.


Reconstructing Open Surfaces via Graph-Cuts.
A novel graph-cuts-based method is proposed for reconstructing open surfaces from unordered point sets. Through a Boolean operation on the crust around the data set, the open surface problem is translated to a watertight surface problem within a restricted region. Integrating the variational model, Delaunay-based tetrahedral mesh and multiphase technique, the proposed method can reconstruct open surfaces robustly and effectively. Furthermore, a surface reconstruction method with domain decomposition is presented, which is based on the new open surface reconstruction method. This method can handle more general surfaces, such as nonorientable surfaces. The algorithm is designed in a parallel-friendly way and necessary measures are taken to eliminate cracks and conflicts between the subdomains. Numerical examples are included to demonstrate the robustness and effectiveness of the proposed method on watertight, open orientable, open nonorientable surfaces and combinations of such.


Suspected aortic dissection and other aortic disorders: multi-detector row CT in 373 cases in the emergency setting.
To retrospectively review the authors' experience with multi-detector row computed tomography (CT) for detection of aortic dissection in the emergency setting. The investigation was institutional review board approved, did not require informed patient consent, and was HIPAA compliant. In 373 clinical evaluations in the emergency setting, 365 patients suspected of having aortic dissection and/or other aortic disorders underwent multidetector CT. Criteria for acute aortic disorder were confirmed by using surgical and pathologic diagnoses or findings at clinical follow-up and any subsequent imaging as the reference standard. Positive cases were characterized according to type of disorder interpreted. Resulting sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated by using two-way contingency tables. All cases found to be negative for acute aortic disorders were grouped according to alternative CT findings. Sixty-seven (18.0%) of the 373 cases were interpreted as positive for acute aortic disorder. One hundred twelve acute aortic disorders were identified in these 67 cases: 23 acute aortic dissections, 14 acute aortic intramural hematomas, 20 acute penetrating aortic ulcers, 44 new or enlarging aortic aneurysms, and 11 acute aortic ruptures. Three hundred five (81.8%) cases were interpreted as negative for acute aortic disorder. In 48 negative cases, multidetector CT depicted alternative findings that accounted for the clinical presentation. Of these, three included both acute aortic disorders and alternative findings, and 45 included only alternative findings. One (0.3%) case was indeterminate for acute aortic disorder. Overall, 112 findings were interpreted as positive for acute aortic disorder, an alternative finding, or both at CT. No interpretations were false-positive, one was false-negative, 67 were true-positive, and 304 were true-negative. Sensitivity, specificity, PPV, NPV, and accuracy were 99% (67 of 68), 100% (304 of 304), 100% (67 of 67), 99.7% (304 of 305), and 99.5% (371 of 373), respectively. The positivity rate for acute aortic dissection or other acute aortic disorder in 373 cases examined at multi-detector row CT was 18.0%.


Parents' and carers' experiences of transition and aftercare following a child's discharge from a paediatric intensive care unit to an in-patient ward setting: A qualitative systematic review.
To explore parents' experiences of transition and aftercare following their child's discharge from a paediatric intensive care unit to an in-patient ward. A qualitative systematic review was conducted. Electronic databases CINAHL, MEDLINE, EMBASE, Psych INFO, and ASSIA were searched for qualitative studies with no date limits imposed. Methodological quality was assessed using the JBI QARI standardised critical appraisal instrument. Data were extracted into a standardised data extraction tool. Findings were pooled using a meta-aggregative approach. Four studies were included in the final review that included a total of 95 participants. Forty-nine findings were extracted and through an iterative process resulting in four synthesised findings being developed. These included: (1) Dynamic emotional response pre, peri and post-transfer; (2) Involvement in care absent but fundamental to functioning; (3) Changes in care delivery and environment provoking adverse emotions and (4) Transition as a physical, emotional and social balancing act. Transitioning from the paediatric intensive care unit to an in-patient ward can be a challenging time for parents, exposing them to a turbulent emotional and social status, and depleting their personal resources. Parents are aware of differences in the organisation and delivery of care between clinical areas which can compound the adversity experienced. Health professionals need to provide targeted support in order to mitigate these negative emotional, physical and social effects experienced.


Barriers and facilitators to ask for lower urinary tract symptoms in people with low back pain and pelvic girdle pain. A qualitative study.
Lower urinary tract symptoms (LUTS) may be a relevant comorbidity when managing people with low back or pelvic girdle pain. It is unknown how often physiotherapists inquire about LUTS, and what the potential barriers and facilitators are to inquire about LUTS in this patient population. To explore the frequency of inquiring about LUTS, and to identify the barriers and facilitators among physiotherapists with and without additional pelvic health training to ask for LUTS in people with low back or pelvic girdle pain. A qualitative study using thematic analysis. Through purposeful sampling, 29 primary care physiotherapists were interviewed (16 physiotherapists and 13 physiotherapists with additional pelvic health training). Thematic analysis was performed to identify themes regarding facilitators and barriers. The frequency of inquiring about LUTS was: 'never': 10%, 'sometimes': 38%, and 'always': 52%. Four barriers were identified: (1) lack of knowledge of the physiotherapist, (2) a standardised assessment approach which did not include LUTS, (3) patient expectations assumed by the physiotherapist, and (4) social, cultural and personal barriers. Three facilitators were identified: (1) communication skills and experience of the physiotherapist, (2) education and knowledge, and (3) interprofessional consultation and referral. The majority of physiotherapists surveyed in this study regularly asked for LUTS in people with low back or pelvic pain. For when not asked, the identified barriers seem modifiable with adequate training, knowledge and skill acquisition, and sound clinical reasoning.


Disseminating Metaproteomic Informatics Capabilities and Knowledge Using the Galaxy-P Framework.
The impact of microbial communities, also known as the microbiome, on human health and the environment is receiving increased attention. Studying translated gene products (proteins) and comparing metaproteomic profiles may elucidate how microbiomes respond to specific environmental stimuli, and interact with host organisms. Characterizing proteins expressed by a complex microbiome and interpreting their functional signature requires sophisticated informatics tools and workflows tailored to metaproteomics. Additionally, there is a need to disseminate these informatics resources to researchers undertaking metaproteomic studies, who could use them to make new and important discoveries in microbiome research. The Galaxy for proteomics platform (Galaxy-P) offers an open source, web-based bioinformatics platform for disseminating metaproteomics software and workflows. Within this platform, we have developed easily-accessible and documented metaproteomic software tools and workflows aimed at training researchers in their operation and disseminating the tools for more widespread use. The modular workflows encompass the core requirements of metaproteomic informatics: (a) database generation; (b) peptide spectral matching; (c) taxonomic analysis and (d) functional analysis. Much of the software available via the Galaxy-P platform was selected, packaged and deployed through an online metaproteomics "Contribution Fest" undertaken by a unique consortium of expert software developers and users from the metaproteomics research community, who have co-authored this manuscript. These resources are documented on GitHub and freely available through the Galaxy Toolshed, as well as a publicly accessible metaproteomics gateway Galaxy instance. These documented workflows are well suited for the training of novice metaproteomics researchers, through online resources such as the Galaxy Training Network, as well as hands-on training workshops. Here, we describe the metaproteomics tools available within these Galaxy-based resources, as well as the process by which they were selected and implemented in our community-based work. We hope this description will increase access to and utilization of metaproteomics tools, as well as offer a framework for continued community-based development and dissemination of cutting edge metaproteomics software.


Classroom-Based Physical Activity and Sedentary Behavior Interventions in Adolescents: A Systematic Review and Meta-Analysis.
It is reported that 81% of adolescents are insufficiently active. Schools play a pivotal role in promoting physical activity (PA) and reducing sedentary behavior (SB). The aim of this systematic review and meta-analysis was to evaluate classroom-based PA and SB interventions in adolescents. A search strategy was developed using the Population Intervention Comparison Outcome Study (PICOS) design framework. Articles were screened using strict inclusion criteria. Study quality was assessed using the Effective Public Health Practice Project quality assessment tool ( http://www.ephpp.ca/tools.html ). Outcome data for preintervention and postintervention were extracted, and effect sizes were calculated using Cohen's d. The strategy yielded 7574 potentially relevant articles. Nine studies were included for review. Study quality was rated as strong for 1 study, moderate for 5 studies, and weak for 3 studies. Five studies were included for meta-analyses, which suggested that the classroom-based interventions had a nonsignificant effect on PA (P = .55, d = 0.05) and a small, nonsignificant effect on SB (P = .16, d = -0.11). Only 9 relevant studies were found, and the effectiveness of the classroom-based PA and SB interventions varied. Based on limited empirical studies, there is not enough evidence to determine the most effective classroom-based methodology to increase PA and SB.


Computational approaches for deciphering the equilibrium and kinetic properties of iron transport proteins.
With the advances in three-dimensional structure determination techniques, high quality structures of the iron transport proteins transferrin and the bacterial ferric binding protein (FbpA) have been deposited in the past decade. These are proteins of relatively large size, and developments in hardware and software have only recently made it possible to study their dynamics using standard computational resources. We review computational techniques towards understanding the equilibrium and kinetic properties of iron transport proteins under different environmental conditions. At the level of detail that requires quantum chemical treatments, the octahedral geometry around iron has been scrutinized and it has been established that the iron coordinating tyrosines are in an unusual deprotonated state. At the atomistic level, both the N-lobe and the full bilobal structure of transferrin have been studied under varying conditions of pH, ionic strength and binding of other metal ions by molecular dynamics (MD) simulations. These studies have allowed questions to be answered, among others, on the function of second shell residues in iron release, the role of synergistic anions in preparing the active site for iron binding, and the differences between the kinetics of the N- and the C-lobe. MD simulations on FbpA have led to the detailed observation of the binding kinetics of phosphate to the apo form, and to the conformational preferences of the holo form under conditions mimicking the environmental niches provided by the periplasmic space. To study the dynamics of these proteins with their receptors, one must resort to coarse-grained methodologies, since these systems are prohibitively large for atomistic simulations. A study of the complex of human transferrin (hTf) with its pathogenic receptor by such methods has revealed a potential mechanistic explanation for the defense mechanism that arises in evolutionary warfare. Meanwhile, the motions in the transferrin receptor bound hTf have been shown to disfavor apo hTf dissociation, explaining why the two proteins remain in complex during the recycling process from the endosome to the cell surface. Open problems and possible technological applications related to metal ion binding-release in iron transport proteins that may be handled by hybrid use of quantum mechanical, MD and coarse-grained approaches are discussed.


Multiethnic Genome-Wide Association Study of Diabetic Retinopathy Using Liability Threshold Modeling of Duration of Diabetes and Glycemic Control.
To identify genetic variants associated with diabetic retinopathy (DR), we performed a large multiethnic genome-wide association study. Discovery included eight European cohorts (n = 3,246) and seven African American cohorts (n = 2,611). We meta-analyzed across cohorts using inverse-variance weighting, with and without liability threshold modeling of glycemic control and duration of diabetes. Variants with a P value <1 × 10-5 were investigated in replication cohorts that included 18,545 European, 16,453 Asian, and 2,710 Hispanic subjects. After correction for multiple testing, the C allele of rs142293996 in an intron of nuclear VCP-like (NVL) was associated with DR in European discovery cohorts (P = 2.1 × 10-9), but did not reach genome-wide significance after meta-analysis with replication cohorts. We applied the Disease Association Protein-Protein Link Evaluator (DAPPLE) to our discovery results to test for evidence of risk being spread across underlying molecular pathways. One protein-protein interaction network built from genes in regions associated with proliferative DR was found to have significant connectivity (P = 0.0009) and corroborated with gene set enrichment analyses. These findings suggest that genetic variation in NVL, as well as variation within a protein-protein interaction network that includes genes implicated in inflammation, may influence risk for DR.


Lotus japonicus cytokinin receptors work partially redundantly to mediate nodule formation.
Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.