Low self-esteem during adolescence predicts poor health, criminal behavior, and limited economic prospects during adulthood.
Using prospective data from the Dunedin Multidisciplinary Health and Development Study birth cohort, the authors found that adolescents with low self-esteem had poorer mental and physical health, worse economic prospects, and higher levels of criminal behavior during adulthood, compared with adolescents with high self-esteem. The long-term consequences of self-esteem could not be explained by adolescent depression, gender, or socioeconomic status. Moreover, the findings held when the outcome variables were assessed using objective measures and informant reports; therefore, the findings cannot be explained by shared method variance in self-report data. The findings suggest that low self-esteem during adolescence predicts negative real-world consequences during adulthood.


Predictors of onset of cannabis and other drug use in male young adults: results from a longitudinal study.
The use of cannabis and other illegal drugs is particularly prevalent in male young adults and is associated with severe health problems. This longitudinal study explored variables associated with the onset of cannabis use and the onset of illegal drug use other than cannabis separately in male young adults, including demographics, religion and religiosity, health, social context, substance use, and personality. Furthermore, we explored how far the gateway hypothesis and the common liability to addiction model are in line with the resulting prediction models. The data were gathered within the Cohort Study on Substance Use Risk Factors (C-SURF). Young men aged around 20 years provided demographic, social, health, substance use, and personality-related data at baseline. Onset of cannabis and other drug use were assessed at 15-months follow-up. Samples of 2,774 and 4,254 individuals who indicated at baseline that they have not used cannabis and other drugs, respectively, in their life and who provided follow-up data were used for the prediction models. Hierarchical logistic stepwise regressions were conducted, in order to identify predictors of the late onset of cannabis and other drug use separately. Not providing for oneself, having siblings, depressiveness, parental divorce, lower parental knowledge of peers and the whereabouts, peer pressure, very low nicotine dependence, and sensation seeking were positively associated with the onset of cannabis use. Practising religion was negatively associated with the onset of cannabis use. Onset of drug use other than cannabis showed a positive association with depressiveness, antisocial personality disorder, lower parental knowledge of peers and the whereabouts, psychiatric problems of peers, problematic cannabis use, and sensation seeking. Consideration of the predictor variables identified within this study may help to identify young male adults for whom preventive measures for cannabis or other drug use are most appropriate. The results provide evidence for both the gateway hypothesis and the common liability to addiction model and point to further variables like depressiveness or practising of religion that might influence the onset of drug use.


Pax6 and eye development in Arthropoda.
The arthropod compound eye is one of the three main types of eyes observed in the animal kingdom. Comparison of the eyes seen in Insecta, Crustacea, Myriapoda and Chelicerata reveals considerable variation in terms of overall cell number, cell positioning, and photoreceptor rhabdomeres, yet, molecular data suggest there may be unexpected similarities. We review here the role of Pax6 in eye development and evolution and the relationship of Pax6 with other retinal determination genes and signaling pathways. We then discuss how the study of changes in Pax6 primary structure, in the gene networks controlled by Pax6 and in the relationship of Pax6 with signaling pathways may contribute to our insight into the relative role of conserved molecular-genetic mechanisms and emergence of evolutionary novelty in shaping the ommatidial eyes seen in the Arthropoda.


Practicing health promotion through pharmacy counseling activities.
Dramatic changes in the U.S. health care system have emphasized the need to promote good health. To achieve this, different types of health care professionals have now started working together. These teams often include participants, such as doctors, pharmacists, and nurses. However, there are many health professionals, such as pharmacists, working in noninstitutionalized settings, such as pharmacies, who are not being fully utilized. One of the ways pharmacists can promote good health is by counseling patients. This article provides some insights regarding the various health promotion activities that are or can be performed by pharmacists. Health promotion educators can play a significant role in educating pharmacists to become effective health promoters. Some hypothetical scenarios and examples, as well as models, are also provided to demonstrate active health promotion through pharmacist counseling activities.


Use of non-invasive ventilation to wean critically ill adults off invasive ventilation: meta-analysis and systematic review.
To summarise the evidence for early extubation with immediate application of non-invasive ventilation compared with continued invasive weaning on important outcomes in intubated adults with respiratory failure. Systematic review and meta-analysis of randomised and quasi-randomised controlled trials. Intensive care units. Critically ill adults receiving invasive ventilation. Study selection criteria We searched Medline, Embase, and CENTRAL, proceedings from four conferences, and reference lists of relevant studies to identify relevant trials. Two reviewers independently selected trials, assessed trial quality, and abstracted data. We identified 12 trials enrolling 530 participants, mostly with chronic obstructive pulmonary disease. Compared with invasive weaning, non-invasive weaning was significantly associated with reduced mortality (relative risk 0.55, 95% confidence interval 0.38 to 0.79), ventilator associated pneumonia (0.29, 95% 0.19 to 0.45), length of stay in intensive care unit (weighted mean difference -6.27 days, -8.77 to -3.78) and hospital (-7.19 days, -10.80 to -3.58), total duration of ventilation, and duration of invasive ventilation. Non-invasive weaning had no effect on weaning failures or weaning time. Benefits on mortality and weaning failures were non-significantly greater in trials that exclusively enrolled patients with chronic obstructive pulmonary disease versus mixed populations. Current trials in critically ill adults show a consistent positive effect of non-invasive weaning on mortality and ventilator associated pneumonia, though the net clinical benefits remain to be fully elucidated. Non-invasive ventilation should preferentially be used in patients with chronic obstructive pulmonary disease in a highly monitored environment.


Probing the exposure of the phosphate group in modified amino acids and peptides by ion-molecule reactions with triethoxyborane in Fourier transform ion cyclotron resonance mass spectrometry.
Intramolecular hydrogen bonds between a phosphate group and charged residues play a crucial role in the chemistry of phosphorylated peptides, driving the species to specific conformations and affecting the exposure of the phosphate moiety. The nature and extent of these interactions can be investigated by measuring the reactivity of phosphate groups toward selected substrates in the gas phase. We used Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry (MS) to perform a systematic study on the gas-phase ionic reactivity of phosphorylated amino acids and peptides with triethoxyborane (TEB). Ions of interest were generated by electrospray ionization (ESI), isolated in the cell of the FT-ICR mass spectrometer, and allowed to react with a stationary pressure of TEB. The temporal evolution of the reaction was monitored and thermal rate constants were derived. The structure of the ionic products was confirmed by Collision-Induced Dissociation (CID) tandem mass spectrometry (MS/MS). TEB was found to react with the phosphate of protonated phosphorylated amino acids and peptides by an addition-elimination pathway. The kinetic efficiency of the reaction showed a positive correlation with the charge state of the reagent ion, suggesting the existence of charge-state-dependent exposure of the phosphate groups towards the incoming neutral during the reaction. Isomeric phosphorylated peptides, only differing for the position of the modified serine residue, showed markedly different kinetic efficiencies. The ability of a phosphorylated species to react with TEB depends on the ease of access to the phosphate moiety in the corresponding gaseous ion. Measuring the kinetic efficiency of such reactions can represent a valuable tool to explore the accessibility of phosphate groups in biomolecules.


Differential proteomic analysis of virus-enriched fractions obtained from plasma pools of patients with dengue fever or severe dengue.
Dengue is the most widespread mosquito-borne viral disease of public health concern. In some patients, endothelial cell and platelet dysfunction lead to life-threatening hemorrhagic dengue fever or dengue shock syndrome. Prognostication of disease severity is urgently required to improve patient management. The pathogenesis of severe dengue has not been fully elucidated, and the role of host proteins associated with viral particles has received little exploration. The proteomes of virion-enriched fractions purified from plasma pools of patients with dengue fever or severe dengue were compared. Virions were purified by ultracentrifugation combined with a water-insoluble polyelectrolyte-based technique. Following in-gel hydrolysis, peptides were analyzed by nano-liquid chromatography coupled to ion trap mass spectrometry and identified using data libraries. Both dengue fever and severe dengue viral-enriched fractions contained identifiable viral envelope proteins and host cellular proteins. Canonical pathway analysis revealed the identified host proteins are mainly involved in the coagulation cascade, complement pathway or acute phase response signaling pathway. Some host proteins were over- or under-represented in plasma from patients with severe dengue compared to patients with dengue fever. ELISAs were used to validate differential expression of a selection of identified host proteins in individual plasma samples of patients with dengue fever compared to patients with severe dengue. Among 22 host proteins tested, two could differentiate between dengue fever and severe dengue in two independent cohorts (olfactomedin-4: area under the curve (AUC), 0.958; and platelet factor-4: AUC, 0.836). A novel technique of virion-enrichment from plasma has allowed to identify two host proteins that have prognostic value for classifying patients with acute dengue who are more likely to develop a severe dengue. The impact of these host proteins on pathogenicity and disease outcome are discussed.


Ethics in psychiatric practice: essential ethics skills, informed consent, the therapeutic relationship, and confidentiality.
Psychiatrists routinely encounter ethical complexities in caring for patients with mental illness, and they are held to the highest levels of accountability in their ethical practices. In this article, the authors first outline skills that are essential to ethical psychiatric practice. They then articulate three domains of clinical and ethical practice that represent the foundation of clinical care for people with mental illness: informed consent, the therapeutic relationship, and confidentiality. Key concepts concerning these domains are presented and relevant empirical evidence concerning each domain is reviewed. An understanding of these clinical and ethical practices will help psychiatrists serve patients with mental illness in their everyday clinical activities in a manner that is respectful, engenders trust, and ultimately fosters optimal clinical care.


Postural orthostatic tachycardia syndrome (POTS): State of the science and clinical care from a 2019 National Institutes of Health Expert Consensus Meeting - Part 1.
Postural orthostatic tachycardia syndrome (POTS) is a chronic and often disabling disorder characterized by orthostatic intolerance with excessive heart rate increase without hypotension during upright posture. Patients often experience a constellation of other typical symptoms including fatigue, exercise intolerance and gastrointestinal distress. A typical patient with POTS is a female of child-bearing age, who often first displays symptoms in adolescence. The onset of POTS may be precipitated by immunological stressors such as a viral infection. A variety of pathophysiologies are involved in the abnormal postural tachycardia response; however, the pathophysiology of the syndrome is incompletely understood and undoubtedly multifaceted. Clinicians and researchers focused on POTS convened at the National Institutes of Health in July 2019 to discuss the current state of understanding of the pathophysiology of POTS and to identify priorities for POTS research. This article, the first of two articles summarizing the information discussed at this meeting, summarizes the current understanding of this disorder and best practices for clinical care. The evaluation of a patient with suspected POTS should seek to establish the diagnosis, identify co-morbid conditions, and exclude conditions that could cause or mimic the syndrome. Once diagnosed, management typically begins with patient education and non-pharmacologic treatment options. Various medications are often used to address specific symptoms, but there are currently no FDA-approved medications for the treatment of POTS, and evidence for many of the medications used to treat POTS is not robust.


Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage-dependent recombination.
Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents.


Bias of least squares approaches for diffusion tensor estimation from array coils in DT-MRI.
Least Squares (LS) and its weighted version are standard techniques to estimate the Diffusion Tensor (DT) from Diffusion Weighted Images (DWI). They require to linearize the problem by computing the logarithm of the DWI. For the single-coil Rician noise model it has been shown that this model does not introduce a significant bias, but for multiple array coils and parallel imaging, the noise cannot longer be modeled as Rician. As a result the validity of LS approaches is not assured. An analytical study of noise statistics for a multiple coil system is carried out, together with the Weighted LS formulation and noise analysis for this model. Results show that the bias in the computation of the components of the DT may be comparable to their variance in many cases, stressing the importance of unbiased filtering previous to DT estimation.


Anti-Inflammatory Effect of Rosa rugosa Flower Extract in Lipopolysaccharide-Stimulated RAW264.7 Macrophages.
Rosa rugosa Thunb, a deciduous shrub of the genus Rosa, has been widely used to treat stomach aches, diarrhoea, pain, and chronic inflammatorydisease in eastern Asia. In recent years, our research team has extensively studied the Rosa rugosa flowerextract, and specificallyundertook pharmacological experiments which have optimized the extraction process. Our methods have yielded a standard extract enriched in phenolic compounds, named PRE. Herein, we expand our efforts and evaluated the antiinflammatoryactivity of PRE on lipopolysaccharide (LPS)-induced inflammationin RAW 264.7 macrophages. PRE significantlyinhibited production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and interleukin 1β (IL-1β), as well as expression of their synthesizing enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase2 (COX-2). Furthermore, PRE inhibited activity of mitogen-activated protein kinases (MAPK) as well as nuclear factor-kappa B (NF-κB) signaling pathway. Our findingsare the firstto explain the anti-inflammatorymechanism by PRE in LPS-stimulated macrophages. Given these results, we propose that PRE has therapeutic potential in the prevention of inflammatory disorders.


Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.
The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.


Optical delay of a signal through a dispersive invisibility cloak.
We present a full-wave analysis method on the transmission of a Gaussian light pulse through a spherical invisibility cloak with causal dispersions. The spatial energy distribution of the Gaussian light pulse is distorted after the transmission. A volcano-shaped spatial time-delay distribution of the transmitted light pulse is demonstrated as a concrete example in our physical model. Both the time-delay and the energy transport depend on the polarization of light waves. This study helps to provide a complete picture of energy propagation through an invisibility cloak.


Corneal endothelial cell density after femtosecond thin-flap LASIK and PRK for myopia: a contralateral eye study.
To compare the effect of femtosecond thinflap LASIK and photorefractive keratectomy (PRK) on postoperative endothelial cell density. In a prospective, randomized, contralateral, single-center clinical trial, 25 patients (mean age: 30+/-5 years [range: 21 to 38 years]) underwent PRK in one eye and thin-flap LASIK in the fellow eye for the correction of myopia using a wavefront-guided platform. The central corneal endothelial cell density was measured using the NIDEK Confoscan 4 preoperatively, and at 1 and 3 months postoperatively. Changes in endothelial cell density were analyzed over time between the two refractive techniques. In PRK, the average preoperative endothelial cell density was 3011+/-329 cells/mm(2), which decreased to 2951+/-327 cells/mm(2) at 1 month (P=.5736) and 2982+/-365 cells/mm(2) at 3 months (P=.6513). In thinflap LASIK, the average preoperative endothelial cell density was 2995+/-325 cells/mm(2), which decreased to 2977+/-358 cells/mm(2) at 1 month (P=.5756) and 2931+/-369 cells/mm(2) at 3 months (P=.4106). No statistically significant difference was found between the two groups at 1 (P=.7404) or 3 (P=.3208) months postoperatively. No statistically significant change was noted in endothelial cell density following either PRK or thin-flap LASIK for the treatment of myopia. Furthermore, no statistically significant difference was found between the two groups out to 3 months postoperatively, indicating that thin-flap LASIK is as safe as PRK with regards to endothelial health.


The role of GLS1-mediated glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals in Th17 responses counteracted by PPARγ agonists.
Background: Peroxisome proliferator-activated receptor gamma (PPARγ) has the ability to counter Th17 responses, but the full mechanisms remain elusive. Herein, we aimed to elucidate this process in view of cellular metabolism, especially glutaminolysis. Methods: MTT, CCK-8, Annexin V-FITC/PI staining or trypan blue exclusion assays were used to analyze cytotoxicity. Flow cytometry and Q-PCR assays were applied to determine Th17 responses. The detection of metabolite levels using commercial kits and rate-limiting enzyme expression using western blotting assays was performed to illustrate the metabolic activity. ChIP assays were used to examine H3K4me3 modifications. Mouse models of dextran sulfate sodium (DSS)-induced colitis and house dust mite (HDM)/lipopolysaccharide (LPS)-induced asthma were established to confirm the mechanisms studied in vitro. Results: The PPARγ agonists rosiglitazone and pioglitazone blocked glutaminolysis but not glycolysis under Th17-skewing conditions, as indicated by the detection of intracellular lactate and α-KG and the fluorescence ratios of BCECF-AM. The PPARγ agonists prevented the utilization of glutamine and thus directly limited Th17 responses even when Foxp3 was deficient. The mechanisms were ascribed to restricted conversion of glutamine to glutamate by reducing the expression of the rate-limiting enzyme GLS1, which was confirmed by GLS1 overexpression. Replenishment of α-KG and 2-HG but not succinate weakened the effects of PPARγ agonists, and α-KG-promoted Th17 responses were dampened by siIDH1/2. Inhibition of KDM5 but not KDM4/6 restrained the inhibitory effect of PPARγ agonists on IL-17A expression, and the H3K4me3 level in the promoter and CNS2 region of the il-17 gene locus down-regulated by PPARγ agonists was rescued by 2-HG and GLS1 overexpression. However, the limitation of PPARγ agonists on the mRNA expression of RORγt was unable to be stopped by 2-HG but was attributed to GSH/ROS signals subsequent to GLS1. The exact role of PPARγ was proved by GW9662 or PPARγ knockout, and the mechanisms for PPARγ-inhibited Th17 responses were further confirmed by GLS1 overexpression in vivo. Conclusion: PPARγ agonists repressed Th17 responses by counteracting GLS1-mediated glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals, which is beneficial for Th17 cell-related immune dysregulation.


Cardiac sympathetic innervation in the MPTP non-human primate model of Parkinson disease.
Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces degeneration of dopaminergic neurons and reproduces the motor features of Parkinson disease (PD); however, the effect of MPTP on extranigral structures has been poorly studied. The aim of this research was to study the cardiac sympathetic innervation of control and MPTP-treated monkeys in order to describe the influence of MPTP toxicity on cardiac tissue. Eight monkeys were included in the study and divided into two groups, four monkeys serving as controls and four forming the MPTP group. Sections from the anterior left ventricle were immunohistochemically examined to characterize the sympathetic fibers of cardiac tissue. The intensity of immunoreactivity in the nerve fibers was quantitatively analyzed using ImageJ software. As occurs in PD, the sympathetic peripheral nervous system is affected in MPTP-treated monkeys. The percentage of tyrosine hydroxylase immunoreactive fibers in the entire fascicle area was markedly lower in the MPTP group (24.23%) than the control group (35.27%) (p < 0.05), with preservation of neurofilament immunoreactive fibers in the epicardium of MPTP-treated monkeys. Alpha-synuclein deposits were observed in sections of the anterior left ventricle of MPTP-treated monkeys but not in control animals, whereas phosphorylated synuclein aggregates were not observed in either controls or MPTP-treated monkeys. The peripheral autonomic system can also be affected by neurotoxins that specifically inhibit mitochondrial complex I.


Characterizing Chain Processes in Visible Light Photoredox Catalysis.
The recognition that Ru(bpy)32+ andsimilar visible light absorbing transition metal complexes can be photocatalysts for a variety of synthetically useful organic reactions has resulted in a recent resurgence of interest in photoredox catalysis. However, many of the critical mechanistic aspects of this class of reactions remain poorly understood. In particular, the degree to which visible light photoredox reactions involve radical chain processes has been a point of some disagreement that has not been subjected to systematic analysis. We have now performed quantum yield measurements to demonstrate that threerepresentative, mechanistically distinct photoredox processes involve product-forming chain reactions. Moreover, we show that the combination of quantum yield and luminescence quenching experiments provides a rapid method to estimate the length of these chains. Together, these measurements constitute a robust, operationally facile strategy for characterizing chain processes in a wide range of visible light photoredox reactions.


Intra-operatively obtained human tissue: protocols and techniques for the study of neural stem cells.
The discoveries of neural (NSCs) and brain tumor stem cells (BTSCs) in the adult human brain and in brain tumors, respectively, have led to a new era in neuroscience research. These cells represent novel approaches to studying normal phenomena such as memory and learning, as well as pathological conditions such as Parkinson's disease, stroke, and brain tumors. This new paradigm stresses the importance of understanding how these cells behave in vitro and in vivo. It also stresses the need to use human-derived tissue to study human disease because animal models may not necessarily accurately replicate the processes that occur in humans. An important, but often underused, source of human tissue and, consequently, both NSCs and BTSCs, is the operating room. This study describes in detail both current and newly developed laboratory techniques, which in our experience are used to process and study human NSCs and BTSCs from tissue obtained directly from the operating room.


Optimal adjuvant therapy for non-small cell lung cancer--how to handle stage I disease.
The standard of care for resected stage II-IIIA non-small cell lung cancer (NSCLC) now includes adjuvant chemotherapy based on the results of three phase III studies using cisplatin-based regimens--the International Adjuvant Lung Trial, the National Cancer Institute of Canada JBR.10 trial, and the Adjuvant Navelbine International Trialist Association trial. The role of adjuvant chemotherapy for stage I disease remains controversial. A recent meta-analysis (the Lung Adjuvant Cisplatin Evaluation) showed potential harm with the addition of adjuvant cisplatin for stage IA disease and no survival benefit for this modality in stage IB disease. Updated results from the Cancer and Leukemia Group B 9633 trial, the only trial to focus exclusively on stage IB patients, no longer show a statistically significant survival benefit from adjuvant chemotherapy in this population, except for the subgroup of patients with larger tumors. It may be that trials have been underpowered to detect a small benefit for patients with stage IB disease, or there may really not be benefit to adding adjuvant therapy for this stage of disease. Additional markers, such as tumor size or the presence or absence of certain tumor proteins like ERCC1, may help to determine which patients with resected stage I NSCLC may benefit from adjuvant chemotherapy. Strategies such as inhibition of angiogenesis pathways and the epidermal growth factor receptor are under exploration.


Testing normative and self-appraisal feedback in an online slot-machine pop-up in a real-world setting.
Over the last few years, there have been an increasing number of gaming operators that have incorporated on-screen pop-up messages while gamblers play on slot machines and/or online as one of a range of tools to help encourage responsible gambling. Coupled with this, there has also been an increase in empirical research into whether such pop-up messages are effective, particularly in laboratory settings. However, very few studies have been conducted on the utility of pop-up messages in real-world gambling settings. The present study investigated the effects of normative and self-appraisal feedback in a slot machine pop-up message compared to a simple (non-enhanced) pop-up message. The study was conducted in a real-world gambling environment by comparing the behavioral tracking data of two representative random samples of 800,000 gambling sessions (i.e., 1.6 million sessions in total) across two conditions (i.e., simple pop-up message versus an enhanced pop-up message). The results indicated that the additional normative and self-appraisal content doubled the number of gamblers who stopped playing after they received the enhanced pop-up message (1.39%) compared to the simple pop-up message (0.67%). The data suggest that pop-up messages influence only a small number of gamblers to cease long playing sessions and that enhanced messages are slightly more effective in helping gamblers to stop playing in-session.


Synthesis and characterization of biocompatible, thermoresponsive ABC and ABA triblock copolymer gelators.
The synthesis of doubly thermoresponsive PPO-PMPC-PNIPAM triblock copolymer gelators by atom transfer radical polymerization using a PPO-based macroinitiator is described. Provided that the PPO block is sufficiently long, dynamic light scattering and differential scanning calorimetry studies confirm the presence of two separate thermal transitions corresponding to micellization and gelation, as expected. However, these ABC-type triblock copolymers proved to be rather inefficient gelators: free-standing gels at 37 degrees C required a triblock copolymer concentration of around 20 wt%. This gelator performance should be compared with copolymer concentrations of 6-7 wt% required for the PNIPAM-PMPC-PNIPAM triblock copolymers reported previously. Clearly, the separation of micellar self-assembly from gel network formation does not lead to enhanced gelator efficiencies, at least for this particular system. Nevertheless, there are some features of interest in the present study. In particular, close inspection of the viscosity vs temperature plot obtained for a PPO43-PMPC160-PNIPAM81 triblock copolymer revealed a local minimum in viscosity. This is consistent with intramicelle collapse of the outer PNIPAM blocks prior to the development of the intermicelle hydrophobic interactions that are a prerequisite for macroscopic gelation.


A novel fifteen minute test for assessment of individual time-dependent clotting responses to aspirin and clopidogrel using modified thrombelastography.
A rapid, reliable, point of care test reflecting patient specific responses to antiplatelet therapy would be of great clinical value in percutaneous coronary intervention (PCI). The aim of this study was to establish whether modified thrombelastography (TEG) can be employed as a 15 minute test of individual patient responses to aspirin and clopidogrel using a novel parameter, percentage clotting inhibition (%CIn). Thirty healthy volunteers and 10 patients undergoing elective PCI were recruited into four groups: 10 volunteers received a single 300 mg dose of aspirin [A1]: 10 volunteers received aspirin 75 mg daily for 7 days [A2]: 10 volunteers received a 600 mg dose of clopidogrel [C1]: 10 patients received a 600 mg loading dose of clopidogrel prior to elective PCI [C2]. In all cases the area under the clotting response curve was measured at 15 minutes (AUC15) and used to calculate a novel parameter, percentage clotting inhibition (%CIn). Large differences were demonstrated in both aspirin and clopidogrel groups in response to therapy as assessed by both the area under the curve at 15 minutes and %CIn. Furthermore, the technique demonstrated important heterogeneity of time-dependent responses between individuals. Modified TEG, employing AUC15 and %CIn, is a promising tool for assessing responses to aspirin and clopidogrel. Further data are now required to assess the potential of this test to optimise individual therapy in PCI patients in order to detect and treat those patients with relative hypo-responsiveness to anti-platelet drugs.


Molecular analysis of a new translocation, t(X;14)(q28;q11), in premalignancy and in leukaemia associated with ataxia telangiectasia.
The disease ataxia telangiectasia (A-T) is a multifaceted disorder in which patients have an increased chance of developing a T-cell leukaemia, often with abnormalities of chromosome 14, but sometimes with rare translocations, like t(X;14)(q28;q11). We describe the cloning of the breakpoint of one such novel t(X;14) from an A-T patient. The translocation breaks within the T cell receptor alpha chain gene on chromosome 14 at band q11 and in a region of the X chromosome, within about 1 Mb of the telomere of the long arm. The patient subsequently developed T-cell prolymphocytic leukaemia (T-PLL), and molecular examination showed that the tumour cells carried the same t(X;14) breakpoint as that cloned from the premalignant cells. The same breakpoint could be detected in blood samples taken as much as 5 years prior to diagnosis of T-PLL. This suggests a role for the abnormality in the tumour development in this patient but implies that other mutational events were necessary for overt disease to become manifest.


How to assure access of essential RMNCH medicines by looking at policy and systems factors: an analysis of countdown to 2015 countries.
In 2000, the Millennium Development Goals set targets for social achievements by 2015 including goals related to maternal and child health, with mixed success. Several initiatives supported these goals including assuring availability of appropriate medicines and commodities to meet health service targets. To reach the new Sustainable Development Goals by 2030, information is needed to address policy and systems factors to improve access to lifesaving commodities. We compiled indicator data on 15 commodities related to reproductive, maternal, newborn, and child health (RMNCH) and analyzed them across 75 Countdown to 2015 countries from eight regions to identify problems with specific commodities and determinants of access. The determinants related to policy, regulatory environment, financing, pharmaceutical procurement and supply chain, and information systems. We mapped commodity information from four datasets from the World Health Organization and the United Nation's Commission on Life Saving Commodities creating a stoplight dashboard to illustrate countries' environment to assure access. We also developed a dashboard for policy and systems indicators for select countries. The commodities we identified as having the fewest barriers to access had been in use longer, including oral rehydration solution and oxytocin injection. Looking across the different systems and policy determinants of access, only Zimbabwe had all 15 commodities on both its essential medicines list and in its standard treatment guidelines, and only Cameroon and Zambia had at least one product registered for each commodity. Senegal alone procured all tracer commodities centrally in the previous year, and 70% of responding countries had costed plans for maternal, newborn, and child health. No country reported recent stock-outs of all the 15 commodities at the central level-countries always had some of the 15 commodities available; however, products with frequent stock-outs included misoprostol, calcium gluconate, penicillin injections, ceftriaxone, and amoxicillin dispersible tablets. This analysis highlights country deficiencies in policies and systems, such as incoherent policy guidelines, problems in product registration, lack of logistics data, and central-level stock-outs that may affect access to essential RMNCH commodities. To tackle these deficiencies, countries need to integrate commodity-related indicators into other health monitoring activities to improve service quality.


Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.
Deficiency of Asparagine Synthetase (ASNSD, MIM 615574) is a very rare autosomal recessive disorder presenting with some brain abnormalities. Affected individuals have congenital microcephaly and progressive encephalopathy associated with severe intellectual disability and intractable seizures. The loss of function of the asparagine synthetase (ASNS, EC 220.127.116.11), particularly in the brain, is the major cause of this particular congenital microcephaly. In this study, we clinically evaluated an affected child from a consanguineous Emirati family presenting with congenital microcephaly and epileptic encephalopathy. In addition, whole-exome sequencing revealed a novel homozygous substitution mutation (c.1193A > C) in the ASNS gene. This mutation resulted in the substitution of highly conserved tyrosine residue by cysteine (p.Y398C). Molecular modeling analysis predicts hypomorphic and damaging effects of this mutation on the protein structure and altering its enzymatic activity. Therefore, we conclude that the loss of ASNS function is most likely the cause of this condition in the studied family. This report brings the number of reported families with this very rare disorder to five and the number of pathogenic mutations in the ASNS gene to four. This finding extends the ASNS pathogenic mutations spectrum and highlights the utility of whole-exome sequencing in elucidation the causes of rare recessive disorders that are heterogeneous and/or overlap with other conditions.


Baseline dependency of minimal clinically important improvement.
Minimal clinically important improvement (MCII) is the smallest outcome measure change important to patients. Research suggests that MCII is dependent on patients' baseline functional status measures. The purposes of this study were: (1) to confirm whether MCII is dependent on patients' admission scores and (2) to test whether MCII is dependent on selected demographic characteristics. This was a prospective, longitudinal, observational cohort study of 6,651 patients with orthopedic knee impairments treated in 332 outpatient rehabilitation clinics in 27 states in the United States. Patient self-reports of functional status (FS) from the Lower Extremity Functional Scale were assessed using a computerized adaptive testing application (0-100 scale). An anchored-based longitudinal method, with a 15-point Likert-type scale (-7 to +7), was used to provide a global rating of change (GROC). The MCII threshold for the GROC was defined at a cut-score of +3 or greater and was determined using nonparametric receiver operating characteristic curve analysis for each of the following variables: sex, symptom acuity, age group, and quartile of baseline FS scores. The results showed that MCII was dependent on patient baseline and demographic characteristics. Patients who were male, were younger, had more-acute symptoms, or had lower FS scores at admission required more FS change to report meaningful change. As this study was a secondary analysis, how the length of treatment mediated the relationship between the independent and dependent variables was unclear. Although a single MCII index may provide a standard cut-score defining the smallest FS change that is meaningful to patients, researchers and clinicians should be aware that MCII is context specific and not a fixed attribute. Current results may help researchers, clinicians, and policy makers to interpret FS change related to the importance of the change to the patient.


Bilateral ischemic optic neuropathy after transurethral prostatic resection: a case report.
Nonarteritic ischemic optic neuropathy affects the anterior portion of the optic nerve and is characterized by sudden, painless visual loss. The affected eye has a relative afferent pupillary defect. The typical funduscopic appearance includes optic disc edema, with associated nerve fiber layer hemorrhage. Risk factors include advanced age, systemic hypertension, nocturnal hypotension, diabetes mellitus, and a small cup-to-disc ratio. Bilateral presentation is rare. Postoperative optic neuropathy has been associated with nonocular surgery; risk factors include a combination of prolonged surgical times, acute systemic hypotension, anemia due to blood loss, or prone positioning. We report for the first time a patient with bilateral, simultaneous anterior ischemic optic neuropathy after elective transurethral prostatic resection. A 66-year old man underwent surgery for benign prostatic hyperplasia. The preoperative blood pressure was 140/85 mmHg, hemoglobin 15.9 g/dL, and hematocrit 48.6%. Two hours postoperatively, the blood pressure, hemoglobin, and hematocrit dropped dramatically. One day later, transient horizontal diplopia developed. Funduscopy showed a congenitally small cup-to-disc ratio without papillary edema. Other ocular findings were unremarkable. By 4 days postoperatively, sudden and painless amaurosis bilaterally developed when the patient awoke with nausea and vomiting. Visual acuity was no light perception bilaterally. The optic discs were swollen with small hemorrhages. Scans of the head and orbits and electrolyte levels were normal. There were no responses on visual evoked potentials bilaterally. The blood pressure was 90/50 mm Hg, the hemoglobin 7.0 g/dL, and the hematocrit 22.9%, necessitating infusion of three units of packed red blood cells. The blood pressure, hematocrit, and hemoglobin increased to normal levels. Three months later the visual acuity remained no light perception. The pupils were unreactive and there was marked optic disc atrophy bilaterally. Bilateral and simultaneous acute ischemic optic neuropathy may be a rare but devastating surgical complication. The combination of anemia and hypotension may increase the risk of anterior ischemic optic neuropathy postoperatively after transurethral prostatic resection.


Replicated anthropogenic hybridisations reveal parallel patterns of admixture in marine mussels.
Human-mediated transport creates secondary contacts between genetically differentiated lineages, bringing new opportunities for gene exchange. When similar introductions occur in different places, they provide informally replicated experiments for studying hybridisation. We here examined 4,279 Mytilus mussels, sampled in Europe and genotyped with 77 ancestry-informative markers. We identified a type of introduced mussels, called "dock mussels," associated with port habitats and displaying a particular genetic signal of admixture between M. edulis and the Mediterranean lineage of M. galloprovincialis. These mussels exhibit similarities in their ancestry compositions, regardless of the local native genetic backgrounds and the distance separating colonised ports. We observed fine-scale genetic shifts at the port entrance, at scales below natural dispersal distance. Such sharp clines do not fit with migration-selection tension zone models, and instead suggest habitat choice and early-stage adaptation to the port environment, possibly coupled with connectivity barriers. Variations in the spread and admixture patterns of dock mussels seem to be influenced by the local native genetic backgrounds encountered. We next examined departures from the average admixture rate at different loci, and compared human-mediated admixture events, to naturally admixed populations and experimental crosses. When the same M. galloprovincialis background was involved, positive correlations in the departures of loci across locations were found; but when different backgrounds were involved, no or negative correlations were observed. While some observed positive correlations might be best explained by a shared history and saltatory colonisation, others are likely produced by parallel selective events. Altogether, genome-wide effect of admixture seems repeatable and more dependent on genetic background than environmental context. Our results pave the way towards further genomic analyses of admixture, and monitoring of the spread of dock mussels both at large and at fine spacial scales.


Targeting metabolic dysregulation for fibrosis therapy.
Fibrosis is the abnormal deposition of extracellular matrix, which can lead to organ dysfunction, morbidity, and death. The disease burden caused by fibrosis is substantial, and there are currently no therapies that can prevent or reverse fibrosis. Metabolic alterations are increasingly recognized as an important pathogenic process that underlies fibrosis across many organ types. As a result, metabolically targeted therapies could become important strategies for fibrosis reduction. Indeed, some of the pathways targeted by antifibrotic drugs in development - such as the activation of transforming growth factor-β and the deposition of extracellular matrix - have metabolic implications. This Review summarizes the evidence to date and describes novel opportunities for the discovery and development of drugs for metabolic reprogramming, their associated challenges, and their utility in reducing fibrosis. Fibrotic therapies are potentially relevant to numerous common diseases such as cirrhosis, non-alcoholic steatohepatitis, chronic renal disease, heart failure, diabetes, idiopathic pulmonary fibrosis, and scleroderma.


SERPINA1 PiZ and PiS heterozygotes and lung function decline in the SAPALDIA cohort.
Severe alpha1-antitrypsin (AAT) deficiency is a strong risk factor for COPD. But the impact of gene variants resulting in mild or intermediate AAT deficiency on the longitudinal course of respiratory health remains controversial. There is indication from experimental studies that pro-inflammatory agents like cigarette smoke can interact with these variants and thus increase the risk of adverse respiratory health effects. Therefore, we tested the effect of the presence of a protease inhibitor (Pi) S or Z allele (PiMS and PiMZ) on the change in lung function in different inflammation-exposed subgroups of a large, population-based cohort study. The SAPALDIA population includes over 4600 subjects from whom SERPINA1 genotypes for S and Z alleles, spirometry and respiratory symptoms at baseline and after 11 years follow-up, as well as proxies for inflammatory conditions, such as detailed smoking history, obesity and high sensitivity C-reactive protein (hs-CRP), were available. All analyses were performed by applying multivariate regression models. There was no overall unfavourable effect of PiMS or PiMZ genotype on lung function change. We found indication that PiZ heterozygosity interacted with inflammatory stimuli leading to an accelerated decline in measures in use as indices for assessing mild airway obstruction. Obese individuals with genotype PiMM had an average annual decline in the forced mid expiratory flow (ΔFEF25-75%) of 58.4 ml whereas in obese individuals with PiMZ it amounted to 92.2 ml (p = 0.03). Corresponding numbers for persistent smokers differed even more strongly (66.8 ml (PiMM) vs. 108.2 ml (PiMZ), p = 0.005). Equivalent, but less strong associations were observed for the change in the FEV1/FVC ratio. We suggest that, in addition to the well established impact of the rare PiZZ genotype, one Z allele may be sufficient to accelerate lung function decline in population subgroups characterized by elevated levels of low grade inflammation.


Phylogenomics of Gesneriaceae using targeted capture of nuclear genes.
Gesneriaceae (ca. 3400 species) is a pantropical plant family with a wide range of growth form and floral morphology that are associated with repeated adaptations to different environments and pollinators. Although Gesneriaceae systematics has been largely improved by the use of Sanger sequencing data, our understanding of the evolutionary history of the group is still far from complete due to the limited number of informative characters provided by this type of data. To overcome this limitation, we developed here a Gesneriaceae-specific gene capture kit targeting 830 single-copy loci (776,754 bp in total), including 279 genes from the Universal Angiosperms-353 kit. With an average of 557,600 reads and 87.8% gene recovery, our target capture was successful across the family Gesneriaceae and also in other families of Lamiales. From our bait set, we selected the most informative 418 loci to resolve phylogenetic relationships across the entire Gesneriaceae family using maximum likelihood and coalescent-based methods. Upon testing the phylogenetic performance of our baits on 78 taxa representing 20 out of 24 subtribes within the family, we showed that our data provided high support for the phylogenetic relationships among the major lineages, and were able to provide high resolution within more recent radiations. Overall, the molecular resources we developed here open new perspectives for the study of Gesneriaceae phylogeny at different taxonomical levels and the identification of the factors underlying the diversification of this plant group.


CT and PET in stomach cancer: preoperative staging and monitoring of response to therapy.
Stomach cancer is one of the leading causes of cancer mortality worldwide. Complete resection of a gastric tumor and adjacent lymph nodes represents the only potentially curative intervention. Computed tomography (CT) has remained the modality of choice for the preoperative staging of gastric cancer and for follow-up. A recently developed advanced CT technique that makes use of thin sections, optimal contrast material enhancement, and multiplanar reformation allows more accurate staging. However, CT may be limited in the identification of nonenlarged lymph node metastasis, peritoneal dissemination, and small hematogenous metastasis. Positron emission tomography (PET) with 2-[fluorine-18]fluoro-2-deoxy-d-glucose (FDG) has been recognized as a useful diagnostic technique in clinical oncology. FDG PET allows scanning of a larger volume than is possible with CT. Although FDG PET is not an appropriate first-line diagnostic procedure in the detection of stomach cancer and is not helpful in tumor staging, it may play a valuable role in the detection of distant metastases, such as those of the liver, lungs, adrenal glands, ovaries, and skeleton. FDG PET may also be helpful in the follow-up of patients undergoing chemotherapy, as it allows the identification of early response to treatment. Further studies are needed to determine the efficacy of FDG PET in the detection of local nodal metastases and peritoneal dissemination. Nevertheless, the combined use of CT and PET can be helpful in the preoperative staging of stomach cancer and in the therapeutic monitoring of affected patients.


The transcriptional regulatory network of proneural glioma determines the genetic alterations selected during tumor progression.
Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression.


Surgeon case volume and 30-day mortality after carotid endarterectomy among contemporary medicare beneficiaries: before and after national coverage determination for carotid artery stenting.
After the 2005 National Coverage Determination to reimburse carotid artery stenting (CAS) for Medicare beneficiaries, the number of CAS procedures increased and carotid endarterectomy (CEA) decreased. We evaluated trends in surgeons' past-year CEA case-volume and 30-day mortality after CEA, and their association before and after the National Coverage Determination. In a retrospective cohort study of patients undergoing CEA (2001-2008) and CAS (2005-2008) using Medicare data, we described yearly trends of CEA and CAS rates, patient characteristics, and 30-day mortality after CEA. We used logistic regression adjusting for patient- and surgeon-level factors to assess the effect of surgeon case volume on 30-day mortality after CEA. We identified 454 717 CEA and 27 943 CAS patients. Patients undergoing CEA in recent years were older and had more comorbidities than earlier years. CEA rates per 10 000 beneficiaries declined from 18.1 in 2002 to 12.7 in 2008, whereas median surgeon past-year case-volume declined from 27 to 21. The CAS rates peaked at 2.3 per 10 000 beneficiaries in 2006 but declined to 1.8 in 2008, resulting in declining overall revascularization procedure rates during 2005 to 2008. Thirty day post-CEA mortality was 1.40% (95% confidence interval, 1.34-1.47) in 2001 to 2002 and 1.17% (1.10-1.24) in 2007 to 2008. Surgeon's past-year case-volume of <10 was associated with higher 30-day mortality consistently during 2001 to 2008. The rate of CEA procedures decreased substantially during 2001 to 2008, as did surgeon past-year case-volume. The postprocedural mortality in Medicare beneficiaries was high compared with trial patients but somewhat improved over time. Those operated by lower past-year case-volume surgeons had increased mortality.


Evaluation of Hair Density in Different Ethnicities in a Healthy American Population Using Quantitative Trichoscopic Analysis.
There is limited research into normative hair density values in individuals of Hispanic descent. In order to help establish baseline density values in these individuals, we evaluated the hair density in 99 Americans of Hispanic descent and compared them with the values in 44 individuals of African descent and 23 Caucasians. Participants were recruited from a New York City medical center and self-identified their ethnicity. Biracial individuals were excluded from the study. The hair density means ± SD ranged from 169 ± 31 to 178 ± 33/cm2 in Americans of Hispanic descent, from 148 ± 25 to 160 ± 27/cm2 in individuals of African descent, and from 214 ± 28 to 230 ± 33/cm2 in Caucasian individuals. All differences in hair densities between the ethnicities were statistically significant (p values ranging from 3.03 × 10-14 to 0.0249). While trichoscopy is not as accurate as invasive histological assessment of hair density or phototrichogram, it is still a reliable and fast method for assessing hair density. Increased awareness of ethnic variances in hair density can help clinicians and researchers diagnose hair disorders, monitor response to therapy, and conduct hair-related research in these patients.


VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae.
Newly synthesized vacuolar hydrolases such as carboxypeptidase Y (CPY) are sorted from the secretory pathway in the late-Golgi compartment and reach the vacuole after a distinct set of membrane-trafficking steps. Endocytosed proteins are also delivered to the vacuole. It has been proposed that these pathways converge at a "prevacuolar" step before delivery to the vacuole. One group of genes has been described that appears to control both of these pathways. Cells carrying mutations in any one of the class E VPS (vacuolar protein sorting) genes accumulate vacuolar, Golgi, and endocytosed proteins in a novel compartment adjacent to the vacuole termed the "class E" compartment, which may represent an exaggerated version of the physiological prevacuolar compartment. We have characterized one of the class E VPS genes, VPS27, in detail to address this question. Using a temperature-sensitive allele of VPS27, we find that upon rapid inactivation of Vps27p function, the Golgi protein Vps10p (the CPY-sorting receptor) and endocytosed Ste3p rapidly accumulate in a class E compartment. Upon restoration of Vps27p function, the Vps10p that had accumulated in the class E compartment could return to the Golgi apparatus and restore correct sorting of CPY. Likewise, Ste3p that had accumulated in the class E compartment en route to the vacuole could progress to the vacuole upon restoration of Vps27p function indicating that the class E compartment can act as a functional intermediate. Because both recycling Golgi proteins and endocytosed proteins rapidly accumulate in a class E compartment upon inactivation of Vps27p, we propose that Vps27p controls membrane traffic through the prevacuolar/endosomal compartment in wild-type cells.


Effects of folate and folylpolyglutamyl synthase modulation on chemosensitivity of breast cancer cells.
Folylpolyglutamyl synthase (FPGS) converts intracellular folates and antifolates to polyglutamates. Polyglutamylated folates and antifolates are retained in cells longer and are better substrates than their monoglutamate counterparts for enzymes involved in one-carbon transfer. FPGS modulation affects the chemosensitivity of cancer cells to antifolates, such as methotrexate, and 5-fluorouracil (5FU) by altering polyglutamylation of antifolates and specific target intracellular folate cofactors. However, this effect may be counterbalanced by FPGS modulation-induced changes in polyglutamylation of other intracellular folate cofactors and total intracellular folate pools. We generated an in vitro model of FPGS overexpression and inhibition in breast cancer cells by stably transfecting human MDA-MB-435 breast cancer cells with the sense FPGS cDNA or FPGS-targeted small interfering RNA, respectively, and investigated the effects of FPGS modulation on chemosensitivity to 5FU and methotrexate. FPGS modulation-induced changes in polyglutamylation of both antifolates and folate cofactors and in intracellular folate pools affected chemosensitivity of breast cancer cells to pemetrexed and trimetrexate whose cytotoxic effects do or do not depend on polyglutamylation, respectively, in a predictable manner. However, the effects of FPGS modulation on the chemosensitivity of breast cancer cells to 5FU and methotrexate seem to be highly complex and depend not only on polyglutamylation of a specific target intracellular folate cofactor or methotrexate, respectively, but also on total intracellular folate pools and polyglutamylation of other intracellular folate cofactors. Whether or not FPGS modulation may be an important clinical determinant of chemosensitivity of breast cancer cells to 5FU and methotrexate-based chemotherapy needs further exploration.


Predictors of adolescent smoking cessation and smoking reduction.
To investigate the processes of change, demographic, health- and smoking-related predictors of both smoking cessation and smoking reduction in adolescents. Data were drawn from a sample of 755 adolescent smokers who participated in a study testing the efficacy of a text messaging-based intervention for smoking cessation. Demographic, health- and smoking-related variables were assessed at baseline. Five processes of smoking cessation, derived from the Transtheoretical Model and the Social Cognitive Theory, as well as outcome measures were assessed at 6-month follow up. Univariate and multivariate regression analyses were conducted to identify baseline and process variables to predict smoking abstinence and smoking reduction. Male gender (OR=0.43, p<.01), lower alcohol consumption (OR=0.90, p=.05) and a lower number of cigarettes smoked per day at baseline (OR=0.87, p<.01) predicted smoking abstinence. Baseline physical activity predicted smoking reduction (OR=1.04, p=.03). None of the examined process variables significantly predicted smoking abstinence. The process variable "counter-conditioning" predicted smoking reduction (OR=1.46, p=.03). Baseline predictors of smoking cessation differ from predictors of smoking reduction. Dynamic or modifiable variables play an important role in predicting adolescent smoking cessation. Counter-conditioning might be an important element in adolescent smoking cessation interventions.


Clustering based on adherence data.
Adherence to a medical treatment means the extent to which a patient follows the instructions or recommendations by health professionals. There are direct and indirect ways to measure adherence which have been used for clinical management and research. Typically adherence measures are monitored over a long follow-up or treatment period, and some measurements may be missing due to death or other reasons. A natural question then is how to describe adherence behavior over the whole period in a simple way. In the literature, measurements over a period are usually combined just by using averages like percentages of compliant days or percentages of doses taken. In the paper we adapt an approach where patient adherence measures are seen as a stochastic process. Repeated measures are then analyzed as a Markov chain with finite number of states rather than as independent and identically distributed observations, and the transition probabilities between the states are assumed to fully describe the behavior of a patient. The patients can then be clustered or classified using their estimated transition probabilities. These natural clusters can be used to describe the adherence of the patients, to find predictors for adherence, and to predict the future events. The new approach is illustrated and shown to be useful with a simple analysis of a data set from the DART (Development of AntiRetroviral Therapy in Africa) trial in Uganda and Zimbabwe.


Search for charged Higgs bosons from top quark decays in pp collisions at square root s=1.96 TeV.
We report the results of a search for a charged Higgs boson in the decays of top quarks produced in pp collisions at a center-of-mass energy of 1.96 TeV. We use a data sample corresponding to an integrated luminosity of 193 pb-1 collected by the upgraded Collider Detector at Fermilab. No evidence for charged Higgs production is found, allowing 95% C.L. upper limits to be placed on BR(t-->H+b) for different charged Higgs decay scenarios. In addition, we present in the minimal supersymmetric standard model (mH+/-, tanbeta) plane the first exclusion regions with radiative and Yukawa coupling corrections.


Varicella-zoster virus gene 66 transcription and translation in latently infected human Ganglia.
Latent infection with varicella-zoster virus (VZV) is characterized by restricted virus gene expression and the absence of virus production. Of the approximately 70 predicted VZV genes, only five (genes 4, 21, 29, 62, and 63) have been shown by multiple techniques to be transcribed during latency. IE62, the protein product of VZV gene 62, is the major immediate-early (IE) virus-encoded transactivator of viral gene transcription and plays a pivotal role in transactivating viral genes during lytic infection. The protein kinase (66-pk) encoded by VZV gene 66 phosphorylates IE62, resulting in cytoplasmic accumulation of IE62 that mitigates nuclear IE62-induced gene activation. Analysis of latently infected human trigeminal ganglia for 66-pk expression by reverse transcriptase-dependent nested PCR, including DNA sequence analysis, in situ hybridization, and immunohistochemistry, revealed VZV open reading frame 66 to be a previously unrecognized latently expressed virus gene and suggests that prevention of IE62 import to the nucleus by VZV 66-pk phosphorylation is one possible mechanism by which VZV latency is maintained.


The impact of vitamin A supplementation on mortality inequalities among children in Nepal.
This paper examines gender, caste and economic differentials in child mortality in the context of a cluster-randomized trial of vitamin A distribution, in order to determine whether or not the intervention narrowed these differentials. The study involved secondary analysis of data from a placebo-controlled randomized field trial of vitamin A supplements. The study took place between 1989-1991 in rural Sarlahi District of Nepal, with 30,059 children age 6 to 60 months. The main outcome measures were differences in mortality between boys and girls, between highest Hindu castes and others, and between the poorest quintile and the four other quintiles. Without vitamin A, girls in rural Nepal experience 26.1 deaths per 1000, which is 8.3 deaths more than the comparison population of boys. With vitamin A the mortality disadvantage of girls is nearly completely attenuated, at only 1.41 additional deaths per 1000 relative to boys. Vitamin A supplementation also narrowed mortality differentials among Hindu castes, but did not lower the concentration of mortality across quintiles of asset ownership. The vitamin A-related attenuation in mortality disadvantage from gender and caste is statistically significant. We conclude that universal supplementation with vitamin A narrowed differentials in child death across gender and caste in rural Nepal. Assuring high-coverage vitamin A distribution throughout Nepal could help reduce inequalities in child survival in this population.


Defining Early Life Stress as a Precursor for Autoimmune Disease.
Childhood exposure to traumatic events, termed early life stress (ELS), is now widely recognized for causing long-term negative health effects that may not manifest until adulthood. Allostatic load (AL) describes the cumulative "wear-and-tear" effects of chronic stress on the body that may adversely affect human health by accelerating other disease processes. Recent epidemiological studies have reported higher stress levels in industrialized countries and trends of increasing prevalence in autoimmune diseases during recent decades. To elucidate mechanisms of stress-related immune dysregulation, most animal studies up to now have focused on AL and stress-triggered events occurring in adults but have not explored ELS in the context of autoimmune disorders. We have identified a current gap in understanding the impact of ELS on immune system ontogeny and its potential for priming genetically susceptible individuals who are at increased risk for autoimmune diseases later in life, through mechanisms involving neuroendocrine-immune cross talk. In this review, we highlight the intersection between stress and immune function, with a focus on ELS as consequential for increased autoimmune disorder risks later in life.


Characterization of an electron conduit between bacteria and the extracellular environment.
A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning beta-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.


Crossing the Divide: Admixture Across the Antarctic Polar Front Revealed by the Brittle Star Astrotoma agassizii.
The Antarctic Polar Front (APF) is one of the most well-defined and persistent oceanographic features on the planet and serves as a barrier to dispersal between the Southern Ocean and lower latitudes. High levels of endemism in the Southern Ocean have been attributed to this barrier, whereas the accompanying Antarctic Circumpolar Current (ACC) likely promotes west-to-east dispersal. Previous phylogeographic work on the brittle star Astrotoma agassizii Lyman, 1875 based on mitochondrial genes suggested isolation across the APF, even though populations in both South American waters and the Southern Ocean are morphologically indistinguishable. Here, we revisit this finding using a high-resolution 2b-RAD (restriction-site-associated DNA) single-nucleotide polymorphism (SNP)-based approach, in addition to enlarged mitochondrial DNA data sets (16S rDNA, COI, and COII), for comparison to previous work. In total, 955 biallelic SNP loci confirmed the existence of strongly divergent populations on either side of the Drake Passage. Interestingly, genetic admixture was detected between South America and the Southern Ocean in five individuals on both sides of the APF, revealing evidence of recent or ongoing genetic contact. We also identified two differentiated populations on the Patagonian Shelf with six admixed individuals from these two populations. These findings suggest that the APF is a strong but imperfect barrier. Fluctuations in location and strength of the APF and ACC due to climate shifts may have profound consequences for levels of admixture or endemism in this region of the world.


IL-1β induces changes in expression of core circadian clock components PER2 and BMAL1 in primary human chondrocytes through the NMDA receptor/CREB and NF-κB signalling pathways.
The circadian clock is a specialised cell signalling circuit present in almost all cells. It controls the timing of key cell activities such as proliferation and differentiation. In osteoarthritis, expression of two components of the circadian clock, BMAL1 and PER2 is altered in chondrocytes and this change has been causally linked with the increase in proliferation and altered chondrocyte differentiation in disease. IL-1β, an inflammatory cytokine abundant in OA joints, has previously been shown to induce changes in BMAL1 and PER2 expression in chondrocytes. The purpose of this study is to identify the mechanism involved. We found IL-1β treatment of primary human chondrocytes led to activation of NMDA receptors as evidenced by an increase in phosphorylation of GluN1 and an increase in intracellular calcium which was blocked by the NMDAR antagonist MK801. Levels of phosphorylated CREB were also elevated in IL-1β treated cells and this effect was blocked by co-treatment of cells with IL-1β and the NMDAR antagonist MK-801. Knockdown of CREB or inhibition of CREB activity prevented the IL-1β induced increase in PER2 expression in chondrocytes but had no effect on BMAL1. Phosphorylated p65 levels were elevated in IL-1β treated chondrocytes indicating increased NF-κB activation. Inhibition of NF-κB activity prevented the IL-1β induced reduction in BMAL1 expression and partially mitigated the IL-1β induced increase in PER2 expression in chondrocytes. These data indicate that the NMDAR/CREB and NF-κB signalling pathways regulate the core circadian clock components PER2 and BMAL1 in chondrocytes. Given that changes in expression of these clock components have been observed in a wide range of diseases, these findings may be broadly relevant for understanding the mechanism leading to circadian clock changes in pathology.


Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae.
Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.


Fluidization of elongated particles-Effect of multi-particle correlations for drag, lift, and torque in CFD-DEM.
Having proper correlations for hydrodynamic forces is essential for successful CFD-DEM simulations of a fluidized bed. For spherical particles in a fluidized bed, efficient correlations for predicting the drag force, including the crowding effect caused by surrounding particles, are already available and well tested. However, for elongated particles, next to the drag force, the lift force, and hydrodynamic torque also gain importance. In this work, we apply recently developed multi-particle correlations for drag, lift and torque in CFD-DEM simulations of a fluidized bed with spherocylindrical particles of aspect ratio 4 and compare them to simulations with widely used single-particle correlations for elongated particles. Simulation results are compared with previous magnetic particle tracking experimental results. We show that multi-particle correlations improve the prediction of particle orientation and vertical velocity. We also show the importance of including hydrodynamic torque.


Spatial and temporal heterogeneity explain disease dynamics in a spatially explicit network model.
There is an increasing recognition that individual-level spatial and temporal heterogeneity may play an important role in metapopulation dynamics and persistence. In particular, the patterns of contact within and between aggregates (e.g., demes) at different spatial and temporal scales may reveal important mechanisms governing metapopulation dynamics. Using 7 years of data on the interaction between the anther smut fungus (Microbotryum violaceum) and fire pink (Silene virginica), we show how the application of spatially explicit and implicit network models can be used to make accurate predictions of infection dynamics in spatially structured populations. Explicit consideration of both spatial and temporal organization reveals the role of each in spreading risk for both the host and the pathogen. This work suggests that the application of spatially explicit network models can yield important insights into how heterogeneous structure can promote the persistence of species in natural landscapes.


Complementary codes for odor identity and intensity in olfactory cortex.
The ability to represent both stimulus identity and intensity is fundamental for perception. Using large-scale population recordings in awake mice, we find distinct coding strategies facilitate non-interfering representations of odor identity and intensity in piriform cortex. Simply knowing which neurons were activated is sufficient to accurately represent odor identity, with no additional information about identity provided by spike time or spike count. Decoding analyses indicate that cortical odor representations are not sparse. Odorant concentration had no systematic effect on spike counts, indicating that rate cannot encode intensity. Instead, odor intensity can be encoded by temporal features of the population response. We found a subpopulation of rapid, largely concentration-invariant responses was followed by another population of responses whose latencies systematically decreased at higher concentrations. Cortical inhibition transforms olfactory bulb output to sharpen these dynamics. Our data therefore reveal complementary coding strategies that can selectively represent distinct features of a stimulus.


Hypersomnia subtypes, sleep and relapse in bipolar disorder.
Though poorly defined, hypersomnia is associated with negative health outcomes and new-onset and recurrence of psychiatric illness. Lack of definition impedes generalizability across studies. The present research clarifies hypersomnia diagnoses in bipolar disorder by exploring possible subgroups and their relationship to prospective sleep data and relapse into mood episodes. A community sample of 159 adults (aged 18-70 years) with bipolar spectrum diagnoses, euthymic at study entry, was included. Self-report inventories and clinician-administered interviews determined features of hypersomnia. Participants completed sleep diaries and wore wrist actigraphs at home to obtain prospective sleep data. Approximately 7 months later, psychiatric status was reassessed. Factor analysis and latent profile analysis explored empirical groupings within hypersomnia diagnoses. Factor analyses confirmed two separate subtypes of hypersomnia ('long sleep' and 'excessive sleepiness') that were uncorrelated. Latent profile analyses suggested a four-class solution, with 'long sleep' and 'excessive sleepiness' again representing two separate classes. Prospective sleep data suggested that the sleep of 'long sleepers' is characterized by a long time in bed, not long sleep duration. Longitudinal assessment suggested that 'excessive sleepiness' at baseline predicted mania/hypomania relapse. This study is the largest of hypersomnia to include objective sleep measurement, and refines our understanding of classification, characterization and associated morbidity. Hypersomnia appears to be comprised of two separate subgroups: long sleep and excessive sleepiness. Long sleep is characterized primarily by long bedrest duration. Excessive sleepiness is not associated with longer sleep or bedrest, but predicts relapse to mania/hypomania. Understanding these entities has important research and treatment implications.


The variability of femoral rotational alignment in total knee arthroplasty.
Several reference axes are used to establish femoral rotational alignment during total knee arthroplasty, but debate continues with regard to which axis is most accurately and easily identified during surgery. Computer-assisted navigation systems have been developed in an attempt to more accurately and consistently align implants during total knee arthroplasty, but it is unknown if navigation systems can improve the accuracy of femoral rotational alignment as compared with that achieved with more traditional techniques involving mechanical guides. The purposes of the present study were to characterize the variability associated with femoral rotational alignment techniques and to determine whether the use of a computer-assisted surgical navigation system reduced this variability. Eleven orthopaedic surgeons used five alignment techniques (including one computer-assisted technique and four traditional techniques) to establish femoral rotational alignment axes on ten cadaveric specimens, and the orientation of these axes was recorded with use of a navigation system. These derived axes were compared against a reference transepicondylar axis on each femur that was established after complete dissection of all soft tissues. There was no difference between the mean errors of all five techniques (p > 0.11). Only 17% of the knees were rotated <5 degrees from the reference transepicondylar axis, with alignment errors ranging from 13 degrees of internal rotation to 16 degrees of external rotation. There were significant differences among the surgeons with regard to their ability to accurately establish femoral rotational alignment axes (p < 0.001). All techniques resulted in highly variable rotational alignment, with no technique being superior. This variability was primarily due to the particular surgeon who was performing the alignment procedure. A navigation system that relies on directly digitizing the femoral epicondyles to establish an alignment axis did not provide a more reliable means of establishing femoral rotational alignment than traditional techniques did.


16S rRNA Amplicon Sequencing Demonstrates that Indoor-Reared Bumblebees (Bombus terrestris) Harbor a Core Subset of Bacteria Normally Associated with the Wild Host.
A MiSeq multiplexed 16S rRNA amplicon sequencing of the gut microbiota of wild and indoor-reared Bombus terrestris (bumblebees) confirmed the presence of a core set of bacteria, which consisted of Neisseriaceae (Snodgrassella), Orbaceae (Gilliamella), Lactobacillaceae (Lactobacillus), and Bifidobacteriaceae (Bifidobacterium). In wild B. terrestris we detected several non-core bacteria having a more variable prevalence. Although Enterobacteriaceae are unreported by non next-generation sequencing studies, it can become a dominant gut resident. Furthermore the presence of some non-core lactobacilli were associated with the relative abundance of bifidobacteria. This association was not observed in indoor-reared bumblebees lacking the non-core bacteria, but having a more standardized microbiota compared to their wild counterparts. The impact of the bottleneck microbiota of indoor-reared bumblebees when they are used in the field for pollination purpose is discussed.


The approach to the Michaelis complex in lactate dehydrogenase: the substrate binding pathway.
We examine here the dynamics of forming the Michaelis complex of the enzyme lactate dehydrogenase by characterizing the binding kinetics and thermodynamics of oxamate (a substrate mimic) to the binary lactate dehydrogenase/NADH complex over multiple timescales, from nanoseconds to tens of milliseconds. To access such a wide time range, we employ standard stopped-flow kinetic approaches (slower than 1 ms) and laser-induced temperature-jump relaxation spectroscopy (10 ns-10 ms). The emission from the nicotinamide ring of NADH is used as a marker of structural transformations. The results are well explained by a kinetic model that has binding taking place via a sequence of steps: the formation of an encounter complex in a bimolecular step followed by two unimolecular transformations on the microsecond/millisecond timescales. All steps are well described by single exponential kinetics. It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His(195) and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster timescales. The on-enzyme K(d) values (the ratios of the microscopic rate constants for each unimolecular step) are not far from one. Either substantial, approximately 10-15%, transient melting of the protein or rearrangements of hydrogen bonding and solvent interactions of a number of water molecules or both appear to take place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes.


Nuclear factor κB up-regulation of CCAAT/enhancer-binding protein β mediates hepatocyte resistance to tumor necrosis factor α toxicity.
The sensitization of hepatocytes to cell death from tumor necrosis factor α (TNFα) underlies many forms of hepatic injury, including that from toxins. Critical for hepatocyte resistance to TNFα toxicity is activation of nuclear factor κB (NF-κB) signaling, which prevents TNFα-induced death by the up-regulation of protective proteins. To further define the mechanisms of hepatocyte sensitization to TNFα killing, immunoblot analysis comparing livers from mice treated with lipopolysaccharide (LPS) alone or LPS together with the hepatotoxin galactosamine (GalN) was performed to identify TNFα-induced protective proteins blocked by GalN. Levels of CCAAT/enhancer-binding protein β (C/EBPβ) were increased after LPS treatment but not GalN/LPS treatment. In a nontransformed rat hepatocyte cell line, TNFα-induced increases in C/EBPβ protein levels were dependent on NF-κB-mediated inhibition of proteasomal degradation. Pharmacological inhibition of c-Jun N-terminal kinase (JNK) did not affect C/EBPβ degradation, indicating that the process was JNK-independent. C/EBPβ functioned to prevent cell death as adenoviral C/EBPβ overexpression blocked TNFα-induced apoptosis in cells sensitized to TNFα toxicity by NF-κB inhibition. C/EBPβ inhibited TNFα-induced caspase 8 activation and downstream mitochondrial cytochrome c release and caspase 3 and caspase 7 activation. Studies in primary hepatocytes from c/ebpβ(-/-) mice confirmed that loss of C/EBPβ increased death from TNFα. c/ebpβ(-/-) mice were also sensitized to liver injury from a nontoxic dose of LPS or TNFα. The absence of jnk2 failed to reverse the GalN-induced block in C/EBPβ induction by LPS, again demonstrating that C/EBPβ degradation was JNK-independent. C/EBPβ is up-regulated by TNFα and mediates hepatocyte resistance to TNFα toxicity by inhibiting caspase-dependent apoptosis. In the absence of NF-κB signaling, proteasomal degradation of C/EBPβ is increased by a JNK-independent mechanism and promotes death from TNFα.


The structure and catalytic mechanism of human sphingomyelin phosphodiesterase like 3a--an acid sphingomyelinase homologue with a novel nucleotide hydrolase activity.
Human sphingomyelinase phosphodiesterase like 3a (SMPDL3a) is a secreted enzyme that shares a conserved catalytic domain with human acid sphingomyelinase (aSMase), the enzyme carrying mutations causative of Niemann-Pick disease. We have solved the structure of SMPDL3a revealing a calcineurin-like fold. A dimetal site, glycosylation pattern and a disulfide bond network are likely to be conserved also in human aSMase. We show that the binuclear site of SMPDL3a is occupied by two Zn(2+) ions and that excess Zn(2+) leads to inhibition of enzyme activity through binding to additional sites. As an extension of recent biochemical work we uncovered that SMPDL3a catalyses the hydrolysis of several modified nucleotides that include cytidine 5'-diphosphocholine, cytidine diphosphate ethanolamine and ADP-ribose, but not the aSMase substrate, sphingomyelin. We subsequently determined the structure of SMPDL3a in complex with the product 5'-cytidine monophosphate (CMP), a structure that is consistent with several distinct coordination modes of the substrate/product in the active site during the reaction cycle. Based on the structure of CMP complexes, we propose a phosphoryl transfer mechanism for SMPDL3a. Finally, a homology model of human aSMase was constructed to allow for the mapping of selected Niemann-Pick disease mutations on a three-dimensional framework to guide further characterization of their effects on aSMase function. Structural data are available in the PDB database under the accession numbers 5EBB and 5EBE.


Ascitic microbiota composition is correlated with clinical severity in cirrhosis with portal hypertension.
Identification of pathogenic bacteria in ascites correlates with poor clinical outcomes. Ascites samples are commonly reported culture-negative, even where frank infection is indicated. Culture-independent methods have previously reported bacterial DNA in ascites, however, whether this represents viable bacterial populations has not been determined. We report the first application of 16S rRNA gene pyrosequencing and quantitative PCR in conjunction with propidium monoazide sample treatment to characterise the viable bacterial composition of ascites. Twenty five cirrhotic patients undergoing paracentesis provided ascites. Samples were treated with propidium monoazide to exclude non-viable bacterial DNA. Total bacterial load was quantified by 16S rRNA Q-PCR with species identity and relative abundance determined by 16S rRNA gene pyrosequencing. Correlation of molecular microbiology data with clinical measures and diagnostic microbiology was performed. Viable bacterial signal was obtained in 84% of ascites samples, both by Q-PCR and pyrosequencing. Approximately 190,000 ribosomal pyrosequences were obtained, representing 236 species, including both gut and non gut-associated species. Substantial variation in the species detected was observed between patients. Statistically significant relationships were identified between the bacterial community similarity and clinical measures, including ascitic polymorphonuclear leukocyte count and Child-Pugh class. Viable bacteria are present in the ascites of a majority of patients with cirrhosis including those with no clinical signs of infection. Microbiota composition significantly correlates with clinical measures. Entry of bacteria into ascites is unlikely to be limited to translocation from the gut, raising fundamental questions about the processes that underlie the development of spontaneous bacterial peritonitis.


Breakthrough trichosporonosis in patients with hematologic malignancies receiving micafungin.
Micafungin is a newly approved antifungal agent in the echinocandin class that is active against Candida species and Aspergillus species. However, this agent has limited activity against a number of fungi, including Trichosporon species. We describe 4 patients who developed disseminated trichosporonosis during the use of micafungin. No cases of trichosporonosis had been seen in the 2 years prior to January 2003, when micafungin became available in our hospital. We reviewed microbiological records of patients at Kameda General Hospital (Kamogawa City, Chiba, Japan) from 1 January 2002 to 31 July 2005, and identified 4 patients whose blood culture results were positive for Trichosporon species. Since January 2003, four patients--3 with acute myelocytic leukemia and 1 with myelodysplastic syndrome--developed disseminated trichosporonosis while receiving treatment with micafungin with or without amphotericin B. The initial 2 isolates were identified as Trichosporon beigelii, and the later 2 isolates were identified as Trichosporon asahii. All 4 patients received micafungin, and 2 also received amphotericin B concomitantly. Minimal inhibitory concentrations of micafungin were >16 microg/mL for the 2 isolates available for susceptibility testing. One patient with hematologic recovery (neutrophils >500 cells/mm3) showed elimination of the fungus after receiving treatment with voriconazole. However, the 3 other patients without hematologic or immunological recovery died of disseminated infection. The rarity of trichosporonosis in our hospital and its emergence after the introduction of micafungin therapy support the idea that micafungin may exert a significant, selective pressure toward resistant fungi, such as Trichosporon species. Therefore, care should be taken regarding the possibility of trichosporonosis in patients receiving micafungin with or without amphotericin B.


Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated.
The major excreted protein (MEP) of transformed mouse fibroblasts is a mannose 6-phosphate-containing glycoprotein whose synthesis and secretion are increased in malignantly transformed 3T3 cells and whose synthesis is increased by treatment of 3T3 cells with tumor promoters or growth factors. When pulse-labeled extracts from Kirsten virus-transformed NIH 3T3 (KNIH) cells were immunoprecipitated using an antibody against secreted MEP, one cellular protein was immunoprecipitated that had the same molecular weight and tryptic peptide map as the secreted protein. Pulse-chase labeling experiments showed that 50-60% of this 39,000-mol-wt form was secreted in transformed cells. Of the 40-50% remaining, approximately 5% was processed into two lower molecular weight forms (29,000 and 20,000) which are sequestered within the cell. Similar processing of these proteins was observed in the nontransformed parent NIH 3T3 (NIH) cells. However, in NIH cells, much less of the synthesized MEP was secreted. Measurements of steady-state levels of these three forms of cellular MEP by Western blot immunolocalization revealed approximately fourfold more MEP in KNIH cells than in NIH cells as well as differences in the relative distribution of MEP forms in transformed and nontransformed cells. Subcellular fractionation of KNIH cells on a Percoll gradient demonstrated a distribution of total MEP similar to that of several lysosomal enzymes. The light lysosomal/Golgi peak from these gradients contained both the precursor 39,000-mol-wt form of MEP and the 20,000-mol-wt form, whereas the heavy lysosomal peak was enriched in the 20,000-mol-wt form. The distribution of MEP forms was found to be similar in NIH cells except that the 29,000-mol-wt form was also seen to be enriched in the heavy lysosomal peak. This biochemical localization of MEP was confirmed by immunolocalization with light and electron microscopy. These data support the hypothesis that MEP is a lysosomal protein that is secreted by transformed cells.


Impact on survival of nuclear atypia in epithelioid malignant mesothelioma.
Malignant pleural mesothelioma is a rare tumour with a bad prognosis. The only consensual prognostic factors are represented by the stage and the histologic type. Concerning the histologic type, epithelioid mesothelioma is known to have better prognosis in comparison with the sarcomatoid and biphasic types. Epithelioid mesotheliomas have been reported to be a heterogeneous prognostic group. Our aim was to assess the impact on the survival of different characteristics of epithelioid mesothelioma, including nuclear atypia, mitotic count, MIB-1 index, inflammatory host response, stromal desmoplasia, necrosis, vascular emboli and invasion depth. We performed a study of survival of 30 malignant pleural mesotheliomas according to the different parameters studied. The study included 26 women and 4 men. The mean age of the patients was 61 years. The microscopic exam concluded to an epithelioid mesothelioma in 17 cases, sarcomatoid mesothelioma in 4 cases and biphasic mesothelioma in 9 cases. The 17 cases of epithelioid mesothelioma developed severe nuclear atypia in 6 cases and mild nuclear atypia in 11 cases. The mitotic count and the MIB-1 score were respectively inferior to 5 mitoses/50 HPF and inferior to 10% in 11 cases and were superior to 5 mitoses/50 HPF and superior to 10% in 6 cases. No vascular emboli were recorded. Tumour necrosis was reported in 1 case. The inflammatory host response was severe in 4 cases and mild in 13 cases. The tumoral stroma was desmoplastic in 4 cases. The invasion depth was superior to 0.5mm in 16 cases. The median overall survival amounted to 180 days. Nuclear atypia was the only feature that had impact on survival in the group of epithelioid mesothelioma. Our results highlight the correlation of nuclear atypia with survival.