Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships.
Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33-35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution.


Clinicopathologic correlation of explanted AlphaCor artificial cornea after exposure of implant.
To describe the clinical presentation and histopathologic findings in a case of explantation of an AlphaCor artificial cornea implant caused by exposure of the skirt. We describe the case report of a 46-year-old man who suffered trauma to the right eye, resulting in 4 failed penetrating keratoplasties (PKPs). Subsequently, an AlphaCor implantation was performed with some visual improvement. Slightly more than 2 years after the implant, skirt exposure occurred, possibly secondary to infectious keratitis in an area of a ruptured bulla, and explantation was performed. Corneal stability was established with repeat corneal transplantation. Histopathologic evaluation of the surgical specimen revealed chronic nongranulomatous inflammation and fibrosis in the peripheral skirt, indicating that biointegration was maintained. However, peripheral corneal stromal melting led to skirt exposure. Focal calcification, as well as retroprosthetic membrane formation, was also identified. The AlphaCor implant is a viable method of treatment for multiple failed PKPs, but it may be associated with unique complications, including corneal stromal melting, focal calcification, and retroprosthetic membrane formation. Infectious keratitis may be a risk factor for corneal stromal melting and needs to be managed aggressively. Explantation of the implant is essential if the skirt is exposed.


Lymphotoxin-Dependent B Cell-FRC Crosstalk Promotes De Novo Follicle Formation and Antibody Production following Intestinal Helminth Infection.
Secondary lymphoid tissues provide specialized niches for the initiation of adaptive immune responses and undergo a remarkable expansion in response to inflammatory stimuli. Although the formation of B cell follicles was previously thought to be restricted to the postnatal period, we observed that the draining mesenteric lymph nodes (mLN) of helminth-infected mice form an extensive number of new, centrally located, B cell follicles in response to IL-4Rα-dependent inflammation. IL-4Rα signaling promoted LTα1β2 (lymphotoxin) expression by B cells, which then interacted with CCL19 positive stromal cells to promote lymphoid enlargement and the formation of germinal center containing B cell follicles. Importantly, de novo follicle formation functioned to promote both total and parasite-specific antibody production. These data reveal a role for type 2 inflammation in promoting stromal cell remodeling and de novo follicle formation by promoting B cell-stromal cell crosstalk.


HB-EGF release mediates glucose-induced activation of the epidermal growth factor receptor in mesangial cells.
Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We showed that transactivation of the epidermal growth factor receptor (EGFR) is an important mediator of matrix upregulation in mesangial cells (MC) in response to high glucose (HG). Here, we study the mechanism of EGFR transactivation. In primary MC, EGFR transactivation by 1 h of HG (30 mM) was unaffected by inhibitors of protein kinase C, reactive oxygen species, or the angiotensin II AT1 receptor. However, general metalloprotease inhibition, as well as specific inhibitors of heparin-binding EGF-like growth factor (HB-EGF), prevented both EGFR and downstream Akt activation. HB-EGF was released into the medium by 30 min of HG, and this depended on metalloprotease activity. One of the metalloproteases shown to cleave proHB-EGF is ADAM17 (TACE). HG, but not an osmotic control, activated ADAM17, and its inhibition prevented EGFR and Akt activation and HB-EGF release into the medium. siRNA to either ADAM17 or HB-EGF prevented HG-induced EGFR transactivation. We previously showed that EGFR/Akt signaling increases transforming growth factor (TGF)-β1 transcription through the transcription factor activator protein (AP)-1. HG-induced AP-1 activation, as assessed by EMSA, was abrogated by inhibitors of metalloproteases, HB-EGF and ADAM17. HB-EGF and ADAM17 siRNA also prevented AP-1 activation. Finally, these inhibitors and siRNA prevented TGF-β1 upregulation by HG. Thus, HG-induced EGFR transactivation in MC is mediated by the release of HB-EGF, which requires activity of the metalloprotease ADAM17. The mechanism of ADAM17 activation awaits identification. Targeting upstream mediators of EGFR transactivation including HB-EGF or ADAM17 provides novel therapeutic targets for the treatment of diabetic nephropathy.


Genetic determinants of cytochrome P450 2A6 activity and biomarkers of tobacco smoke exposure in relation to risk of lung cancer development in the Shanghai cohort study.
Cytochrome P450 2A6 (CYP2A6) catalyzes nicotine metabolism and contributes to the metabolism of the tobacco-specific lung carcinogen, NNK. Genetic variation in CYP2A6 may affect smoking behavior and contribute to lung cancer risk. A nested case-control study of 325 lung cancer cases and 356 controls was conducted within a prospective cohort of 18,244 Chinese men in Shanghai, China. Quantified were 4 allelic variants of CYP2A6 [*1(+51A), *4, *7, and *9] and urinary total nicotine, total cotinine, total trans-3'-hydroxycotinine (3HC) and total NNAL (an NNK metabolite). Calculated were total nicotine equivalents (TNE), the sum of total nicotine, total cotinine and total 3HC and the total 3HC:total cotinine ratio as a measure of CYP2A6 activity. The nicotine metabolizer status (normal, intermediate, slow and poor) was determined by CYP2A6 genotypes. The smoking-adjusted odds ratios (95% confidence intervals) of lung cancer for the highest vs lowest quartile of total nicotine, total cotinine, total 3HC, TNE and total NNAL were 3.03 (1.80-5.10), 4.70 (2.61-8.46), 4.26 (2.37-7.68), 4.71 (2.61-8.52), and 3.15 (1.86-5.33) (all Ptrend < 0.001), respectively. Among controls CYP2A6 poor metabolizers had a 78% lower total 3HC:total cotinine ratio and 72% higher total nicotine (Ptrend ≤ 0.002). Poor metabolizers had an odds ratio of 0.64 (95% confidence interval = 0.43-0.97) for lung cancer, which was statistically nonsignificant (odds ratio = 0.74, 95% confidence interval = 0.48-1.15) after adjustment for urinary TNE and smoking intensity and duration. The lower lung cancer risk observed in CYP2A6 poor metabolizers is partially explained by the strong influence of CYP2A6 genetic polymorphisms on nicotine uptake and metabolism.


GH-dependent growth of experimentally induced carcinomas in vivo.
Interest in investigating the role of the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis in the initiation and progression of experimentally induced carcinomas has arisen due to several observations in the human population. First, subjects with Laron syndrome who lack GH signaling have significantly lower rates of cancer than people who have normal GH signaling. Second, epidemiologic studies have found strong associations between elevated circulating IGF-1 and the incidence of several common cancers. Third, women who bear children early in life have a dramatically reduced risk of developing breast cancer, which may be due to differences in hormone levels including GH. These observations have motivated multiple studies that have experimentally altered activity of the GH/IGF-1 axis in the context of experimental carcinoma models in mice and rats. Most of these studies have utilized carcinoma models for four organ systems that are also frequent sites of carcinomas in humans: the mammary gland, prostate gland, liver, and colon. This review focuses on these studies and describes some of the most common genetic models used to alter the activity of the GH/IGF-1 axis in experimentally induced carcinomas. A recurring theme that emerges from these studies is that manipulations that reduce the activity of GH or mediators of GH action also inhibit carcinogenesis in multiple model systems.


Heart rate recovery and heart rate complexity following resistance exercise training and detraining in young men.
The purpose of this study was to examine heart rate recovery (HRR) and linear/nonlinear heart rate variability (HRV) before and after resistance training. Fourteen young men (25.0 +/- 1.1 yr of age) completed a crossover design consisting of a 4-wk time-control period, 6 wk of resistance training (3 days/wk), and 4 wk of detraining. Linear HRV was spectrally decomposed using an autoregressive approach. Nonlinear dynamics of heart rate complexity included sample entropy (SampEn) and Lempel-Ziv entropy (LZEn). HRR was calculated from a graded maximal exercise test as maximal heart rate attained during the test minus heart rate at 1 min after exercise (HRR). There was no change in SampEn, LZEn, or HRR after the time-control portion of the study (P > 0.05). SampEn (P < 0.05), LZEn (P < 0.05), and HRR (P < 0.05) increased after resistance training and returned to pretraining values after detraining. There was no change in spectral measures of HRV at any time point (P > 0.05). These findings suggest that resistance exercise training increases heart rate complexity and HRR after exercise but has no effect on spectral measures of HRV in young healthy men. These autonomic changes regress shortly after cessation of training.


Multicenter, double-blind, randomized, phase II trial to assess the safety and efficacy of ceftolozane-tazobactam plus metronidazole compared with meropenem in adult patients with complicated intra-abdominal infections.
Ceftolozane-tazobactam (TOL-TAZ) is a novel antibacterial with activity against Pseudomonas aeruginosa and other common Gram-negative pathogens, including extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, that are associated with complicated intra-abdominal infections (cIAIs). This prospective, double-blind, randomized, multicenter, phase II trial assessed patient clinical and microbiological responses to and the safety of TOL-TAZ plus metronidazole compared with those of meropenem. Hospitalized adults with cIAIs that required surgical intervention were randomized (2:1) to receive intravenous (i.v.) TOL-TAZ (1.5 g [containing 1,000 mg TOL and 500 mg TAZ] every 8 h [q8h]) with or without i.v. metronidazole (500 mg q8h) or i.v. meropenem (1 g q8h) for 4 to 7 days. The primary endpoint was the clinical response at the test-of-cure visit in the microbiologically modified intent-to-treat (mMITT) and microbiologically evaluable (ME) populations. Secondary measures included the patients' microbiological response and safety. In total, 82 patients received TOL-TAZ (90.2% with metronidazole), and 39 received meropenem. For the mMITT population, clinical cure was seen in 83.6% of the patients (51/61; 95% confidence interval [CI], 71.9 to 91.8) who received TOL-TAZ and 96.0% of the patients (24/25; 95% CI, 79.6 to 99.9) who received meropenem (difference, -12.4%; 95% CI, -34.9% to 11.1%); in the ME population, clinical cure was seen in 88.7% and 95.8% of the patients (difference, -7.1%; 95% CI, -30.7% to 16.9%) who received TOL-TAZ and meropenem, respectively. TOL-TAZ demonstrated microbiological success against Escherichia coli (89.5%), Klebsiella pneumoniae (100%), and P. aeruginosa (100%). The adverse event rates were similar in the groups (50.0% with TOL-TAZ and 48.8% with meropenem). TOL-TAZ in combination with metronidazole was well tolerated and resulted in clinical and microbiological success rates supportive of further clinical development in patients with cIAIs. (This study has been registered at ClinicalTrials.gov under registration no. NCT01147640.).


Parameterized Analysis of Multiobjective Evolutionary Algorithms and the Weighted Vertex Cover Problem.
Evolutionary multiobjective optimization for the classical vertex cover problem has been analysed in Kratsch and Neumann (2013) in the context of parameterized complexity analysis. This article extends the analysis to the weighted vertex cover problem in which integer weights are assigned to the vertices and the goal is to find a vertex cover of minimum weight. Using an alternative mutation operator introduced in Kratsch and Neumann (2013), we provide a fixed parameter evolutionary algorithm with respect to OPT, the cost of an optimal solution for the problem. Moreover, we present a multiobjective evolutionary algorithm with standard mutation operator that keeps the population size in a polynomial order by means of a proper diversity mechanism, and therefore, manages to find a 2-approximation in expected polynomial time. We also introduce a population-based evolutionary algorithm which finds a (1+ɛ)-approximation in expected time O(n·2min{n,2(1-ɛ)OPT}+n3).


Ten-year predicted coronary heart disease risk in HIV-infected men and women.
Highly active antiretroviral therapy (HAART), in addition to traditional vascular risk factors, may affect coronary heart disease (CHD) risk in individuals with human immunodeficiency virus (HIV) infection. Among HIV-infected (931 men and 1455 women) and HIV-uninfected (1099 men and 576 women) adults, the predicted risk of CHD was estimated on the basis of age, sex, lipid and blood pressure levels, the presence of diabetes, and smoking status. Among HIV-infected men, 2% had moderate predicted risk of CHD (10-year CHD risk, 15%-25%), and 17% had high predicted risk (10-year CHD risk of > or = 25% or diabetes). Among HIV-infected women, 2% had moderate predicted CHD risk, and 12% had high predicted CHD risk. Compared with users of protease inhibitor-based HAART, the adjusted odds ratio (OR) for moderate-to-high risk of CHD was significantly lower among HAART-naive individuals (OR, 0.57; 95% confidence interval [CI], 0.36-0.89). Users of HAART that was not protease inhibitor based (OR, 0.74; 95% CI, 0.53-1.01) and former HAART users (OR, 0.68; 95% CI, 0.46-1.03) were also less likely than users of protease inhibitor-based HAART to have moderate-to-high CHD risk, although 95% CIs overlapped the null. Low income was associated with increased likelihood of moderate-to-high CHD risk (for annual income < $10,000 vs. > $40,000: OR, 2.32; 95% CI, 1.51-3.56 ). Elevated body mass index (calculated as weight in kilograms divided by the square of height in meters) predicted increased likelihood of moderate-to-high CHD risk (for BMI of 18.5-24.9 vs. BMI of 25-30: OR, 1.41 [95% CI, 1.03-1.93]; for BMI of 18.5-24.9 vs. BMI > or = 30: OR, 1.79 [95% CI, 1.25-2.56]). Among HIV-infected adults, in addition to antiretroviral drug exposures, being overweight and having a low income level were associated with increased predicted CHD risk. This suggests a need to target HIV-infected men and women with these characteristics for vascular risk factor screening.


Suppression of microRNA activity amplifies IFN-γ-induced macrophage activation and promotes anti-tumour immunity.
Tumour-associated macrophages (TAMs) largely express an alternatively activated (or M2) phenotype, which entails immunosuppressive and tumour-promoting capabilities. Reprogramming TAMs towards a classically activated (M1) phenotype may thwart tumour-associated immunosuppression and unleash anti-tumour immunity. Here we show that conditional deletion of the microRNA (miRNA)-processing enzyme DICER in macrophages prompts M1-like TAM programming, characterized by hyperactive IFN-γ/STAT1 signalling. This rewiring abated the immunosuppressive capacity of TAMs and fostered the recruitment of activated cytotoxic T lymphocytes (CTLs) to the tumours. CTL-derived IFN-γ exacerbated M1 polarization of Dicer1-deficient TAMs and inhibited tumour growth. Remarkably, DICER deficiency in TAMs negated the anti-tumoral effects of macrophage depletion by anti-CSF1R antibodies, and enabled complete tumour eradication by PD1 checkpoint blockade or CD40 agonistic antibodies. Finally, genetic rescue of Let-7 miRNA activity in Dicer1-deficient TAMs partly restored their M2-like phenotype and decreased tumour-infiltrating CTLs. These findings suggest that DICER/Let-7 activity opposes IFN-γ-induced, immunostimulatory M1-like TAM activation, with potential therapeutic implications.


Exercise Oscillatory Ventilation: Interreviewer Agreement and a Novel Determination.
Determination of exercise oscillatory ventilation (EOV) is subjective, and the interreviewer agreement has not been reported. The purposes of this study were, among patients with heart failure (HF), as follows: 1) to determine the interreviewer agreement for EOV and 2) to describe a novel, objective, and quantifiable measure of EOV. This was a secondary analysis of the HEART Camp: Promoting Adherence to Exercise in Patients with Heart Failure study. EOV was determined through a blinded review by six individuals on the basis of their interpretation of the EOV literature. Interreviewer agreement was assessed using Fleiss kappa (κ). Final determination of EOV was based on agreement by four of the six reviewers. A new measure (ventilation dispersion index; VDI) was calculated for each test, and its ability to predict EOV was assessed with the receiver operator characteristics curve. Among 243 patients with HF (age, 60 ± 12 yr; 45% women), the interreviewer agreement for EOV was fair (κ = 0.303) with 10-s discrete data averages and significantly better, but only moderate (κ = 0.429) with 30-s rolling data averages. Prevalence rates of positive and indeterminate EOVs were 18% and 30% with the 10-s discrete averages and 14% and 13% with the 30-s rolling averages, respectively. VDI was strongly associated with EOV, with areas under the receiver operator characteristics curve of 0.852 to 0.890. Interreviewer agreement for EOV in patients with HF is fair to moderate, which can negatively affect risk stratification. VDI has strong predictive validity with EOV; as such, it might be a useful measure of prognosis in patients with HF.


Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers.
Development of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes. We searched for markers using the differential in-gel electrophoresis proteomic technology to compare proteins of bovine nucleus pulposus cells, phenotypically similar to mature human nucleus cells, with those of bovine articular chondrocytes. In the cohort of the differentially expressed proteins identified by mass spectrometry, cytokeratin 8 (CK8) was further validated by immunostaining of freshly isolated cells and frozen tissue sections using monoclonal antibodies. We identified a set of 14 differentially expressed proteins. Immunohistochemistry showed that only a subset of cells (approximately 10%) was positive for one of these proteins, CK8, an intermediate filament protein present in epithelial but not mesenchymal cells. In tissue sections, CK8-positive cells were seen in all discs examined and appeared as small isolated clusters surrounded by gelatinous matrix. Notochordal nucleus pulposus cells from pig, phenotypically similar to human infant nucleus pulposus cells, were all CK8-positive. The mesenchymal intermediate filament protein vimentin was present in all bovine and porcine nucleus pulposus cells. The notochordal cell population is reported to disappear from the nucleus pulposus of bovine discs before birth and from human discs in childhood. However our finding of the co-expression of vimentin and CK8 in small isolated clusters of the bovine nucleus pulposus cells indicates that a subpopulation of notochordal-like cells remains in the mature bovine disc. This finding agrees with reports in the literature on co-expression of cytokeratins and vimentin in adult human discs. As notochordal cells produce factors that promote matrix production, the CK8-positive subpopulation could have important implications for activity and survival of the nucleus pulposus, and should be considered in development of cell therapies for disc repair. In addition, the finding of differential expression of proteins in the cell population of nucleus pulposus has implications with regard to the search for specific markers.


Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis.
Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglutathione, which in turn is converted to D-lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo-transfected control cells (21.80+/-0.1 vs. 0. 76+/-0.02 micromol/min/mg protein, n = 3, P < 0.001). In neo-transfected cells, 30 mM glucose incubation increased MG and D-lactate concentration approximately twofold above 5 MM (35.5+/-5.8 vs. 19.6+/-1.6, P < 0.02, n = 3, and 21.0+/-1.3 vs. 10.0+/-1.2 pmol/ 10(6) cells, n = 3, P < 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration at all, while increasing the enzymatic product D-lactate by > 10-fold (18.9+/-3.2 vs. 18.4+/- 5.8, n = 3, P = NS, and 107.1+/-9.0 vs. 9.4+/-0 pmol/10(6) cells, n = 3, P < 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58+/-0.15 vs. 0.19+/-0.03 total absorbance units, n = 3, P < 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis.


Crystallization and preliminary diffraction studies of the extracellular region of human p58 killer cell inhibitory receptor (KIR2).
Molecules of the human killer cell inhibitory receptor (KIR) family, which belong to the immunoglobulin superfamily (IgSF), are expressed on the surface of natural killer (NK) cells and some subsets of T cells. These receptors function to mediate the inhibition or activation of cytotoxic activity by recognizing HLA class I molecules on the target cell. The extracellular region of a p58 KIR specific for HLA-Cw1,3,7 (KIR2) has been overproduced in Escherichia coli and purified. The recombinant KIR2 has been crystallized in 9-10% poly(ethylene glycol) methyl ether (average Mr = 8000), 50mM HEPES, 8% ethylene glycol, 0.5% octyl-beta-glucoside, pH 7.5, at 294 K using the sitting-drop vapour-diffusion method. Preliminary X-ray diffraction studies reveal the space group to be hexagonal (P6122 or P6522) with lattice constants a = b = 95.3, c = 130.8 A. A native data set (3 A resolution) has been collected at the Photon Factory (lambda = 1.0 A).


RF-MaloSite and DL-Malosite: Methods based on random forest and deep learning to identify malonylation sites.
Malonylation, which has recently emerged as an important lysine modification, regulates diverse biological activities and has been implicated in several pervasive disorders, including cardiovascular disease and cancer. However, conventional global proteomics analysis using tandem mass spectrometry can be time-consuming, expensive and technically challenging. Therefore, to complement and extend existing experimental methods for malonylation site identification, we developed two novel computational methods for malonylation site prediction based on random forest and deep learning machine learning algorithms, RF-MaloSite and DL-MaloSite, respectively. DL-MaloSite requires the primary amino acid sequence as an input and RF-MaloSite utilizes a diverse set of biochemical, physiochemical and sequence-based features. While systematic assessment of performance metrics suggests that both 'RF-MaloSite' and 'DL-MaloSite' perform well in all metrics tested, our methods perform particularly well in the areas of accuracy, sensitivity and overall method performance (assessed by the Matthew's Correlation Coefficient). For instance, RF-MaloSite exhibited MCC scores of 0.42 and 0.40 using 10-fold cross-validation and an independent test set, respectively. Meanwhile, DL-MaloSite was characterized by MCC scores of 0.51 and 0.49 based on 10-fold cross-validation and an independent set, respectively. Importantly, both methods exhibited efficiency scores that were on par or better than those achieved by existing malonylation site prediction methods. The identification of these sites may also provide important insights into the mechanisms of crosstalk between malonylation and other lysine modifications, such as acetylation, glutarylation and succinylation. To facilitate their use, both methods have been made freely available to the research community at https://github.com/dukkakc/DL-MaloSite-and-RF-MaloSite.


HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins.
Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.


School polices, programmes and facilities, and objectively measured sedentary time, LPA and MVPA: associations in secondary school and over the transition from primary to secondary school.
There is increasing policy interest in ensuring that the school environment supports healthy behaviours. We examined the cross-sectional and longitudinal associations between schools' policies, programmes and facilities for physical activity (PA) and adolescents' objectively-measured activity intensity during the school day and lunchtime. Accelerometer-derived PA (proportion of time spent in sedentary (SED), light PA (LPA) and moderate-to-vigorous PA (MVPA)) during school hours and lunchtime from 325 participants in the SPEEDY study were obtained from baseline measurements (primary school, age 9/10 years) and +4y follow-up (secondary school). School environment characteristics were assessed by teacher questionnaire. Multivariable multi-level linear regression analyses accounting for school and adjusted for sex, age, BMI and family socio-economic status assessed cross-sectional associations with lunchtime and school-day SED, LPA and MVPA; effect modification by sex was investigated. The association of changes in school environment with changes in outcomes was examined using multivariable cross-classified linear regression models. There were significant differences between primary and secondary schools for 6/10 school environment characteristics investigated (including secondary schools reporting shorter breaks, more lunchtime PA opportunities, and higher number of sports facilities). Cross-sectional analyses showed that boys attending secondary schools with longer breaks spent significantly less time in SED and more time in MVPA during the school day. Longitudinally, an increase in break-time duration between primary and secondary school was associated with smaller reductions in MVPA during the school day. Moreover, participants who moved from a primary school that did not provide opportunities for PA at lunchtime to a secondary school that did provide such opportunities exhibited smaller increases in SED and smaller reductions in MVPA at lunchtime. Schools should consider the potential negative impact of reducing break time duration on students' MVPA and SED during the school day. School-based interventions that combine longer breaks and more PA opportunities during lunchtime may be a fruitful direction for future research. Further research should also explore other factors in the school environment to explain the school-level clustering observed, and study sex differences in the way that the school environment influences activity intensity for adolescent populations.


Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei x C57BL/6J).
Strain CAST/Ei (CAST) mice exhibit unusually low levels of high density lipoproteins (HDL) as compared with most other strains of mice, including C57BL/6J (B6). This appears to be due in part to a functional deficiency of lecithin:cholesterol acyltransferase (LCAT). LCAT mRNA expression in CAST mice is normal, but the mice exhibit several characteristics consistent with functional deficiency. First, the activity and mass of LCAT in plasma and in HDL of CAST mice were reduced significantly. Second, the HDL of CAST mice were relatively poor in phospholipids and cholesteryl esters, but rich in free cholesterol and apolipoprotein A-I (apoA-I). Third, the adrenals of CAST mice were depleted of cholesteryl esters, a phenotype similar to that observed in LCAT- and acyl-CoA:cholesterol acyltransferase-deficient mice. Fourth, in common with LCAT-deficient mice, CAST mice contained triglyceride-rich lipoproteins with "panhandle"-like protrusions. To examine the genetic bases of these differences, we studied HDL lipid levels in an intercross between strain CAST and the common laboratory strain B6 on a low fat, chow diet as well as a high fat, atherogenic diet. HDL levels exhibited complex inheritance, as 12 quantitative trait loci with significant or suggestive likelihood of observed data scores were identified. Several of the loci occurred over plausible candidate genes and these were investigated. The results indicate that the functional LCAT deficiency is unlikely to be due to variations of the LCAT gene. Our results suggest that novel genes are likely to be important in the control of HDL metabolism, and they provide evidence of genetic factors influencing the interaction of LCAT with HDL.


Preparation and purification of synthetic protein nanoparticulates.
The protein nanostructure used in this study (bovine serum albumin; BSA nanoparticles) were fabricated with an average nanoparticle diameter 150 nm based on the principle of coacervation. Practical recovery of nanoparticulate mimics, of products such as plasmid DNA and viruses as putative gene therapy vectors from model systems, has been studied. The adsorbents employed in this study for the recovery of nanoparticles had one of four discrete designs i.e. microporous (pore size <0.2 microm), macroporous (pore size >0.8 microm), solid phase (nonporous) and pellicular (pore size <0.5 microm). Soluble protein was included in the study to represent cellular components of complex feedstocks and the separation of assemblies from components, while particulate protein served as surrogate size and charge mimics of less easily sourced viral and plasmid gene therapy vectors. Candidate adsorbents were physically characterised to assess their suitability for fluidised-bed operation, biochemically characterised exploiting batch-binding experimentation and laser scanning confocal microscopy. The adsorptive capacity of nanoparticulate products was strongly influenced by the physical design of the adsorbents, and microporous adsorbents appeared to be less suited for the recovery of nanoparticulate products. The generic application of such adsorbents for the recovery of nanoparticulate bioproducts is discussed.


Treatment strategy for rod fractures following corrective fusion surgery in adult spinal deformity depends on symptoms and local alignment change.
OBJECTIVE Despite the significant incidence of rod fractures (RFs) following long-segment corrective fusion surgery, little is known about the optimal treatment strategy. The objectives of this study were to investigate the time course of clinical symptoms and treatments in patients with RFs following adult spinal deformity (ASD) surgery and to establish treatment recommendations. METHODS This study was a retrospective case series of patients with RFs whose data were retrieved from a prospectively collected single-center database. The authors reviewed the cases of 304 patients (mean age 62.9 years) who underwent ASD surgery. Primary symptoms, time course of symptoms, and treatments were investigated by reviewing medical records. Standing whole-spine radiographs obtained before and after RF development and at last follow-up were evaluated. Osseous union was assessed using CT scans and intraoperative findings. RESULTS There were 54 RFs in 53 patients (mean age 68.5 years [range 41-84 years]) occurring at a mean of 21 months (range 6-47 months) after surgery. In 1 patient RF occurred twice, with each case at a different time and level, and the symptoms and treatments for these 2 RFs were analyzed separately (1 case of revision surgery and 1 case of nonoperative treatment). The overall rate of RF observed on radiographs after a minimum follow-up of 1 year was 18.0% (54 of 300 cases). The clinical symptoms at the time of RF were pain in 77.8% (42 of 54 cases) and no onset of new symptoms in 20.5% (11 of 54 cases). The pain was temporary and had subsided in 19 of 42 cases (45%) within 2 weeks. In 36 of the 54 cases (66.7%) (including the first RF in the patient with 2 RFs), patients underwent revision surgery at a mean of 116 days (range 5-888 days) after diagnosis. In 18 cases patients received only nonoperative treatment as of the last follow-up, including 17 cases in which the patients experienced no pain and no remarkable progression of deformity (mean 18.5 months after RF development). CONCLUSIONS This analysis of 54 RFs in 53 patients following corrective fusion surgery for ASD demonstrates a relationship between symptoms and alignment change. Revision surgeries were performed in a total of 36 cases. Nonoperative care was offered in 18 (33.3%) of 54 cases at the last follow-up, with no additional symptoms in 17 of the 18 cases. These data offer useful information regarding informed decision making for patients in whom an RF occurs after ASD surgery.


Understanding and improving global crop response to ozone pollution.
Concentrations of ground-level ozone ([O3 ]) over much of the Earth's land surface have more than doubled since pre-industrial times. The air pollutant is highly variable over time and space, which makes it difficult to assess the average agronomic and economic impacts of the pollutant as well as to breed crops for O3 tolerance. Recent modeling efforts have improved quantitative understanding of the effects of current and future [O3 ] on global crop productivity, and experimental advances have improved understanding of the cellular O3 sensing, signaling and response mechanisms. This work provides the fundamental background and justification for breeding and biotechnological approaches for improving O3 tolerance in crops. There is considerable within-species variation in O3 tolerance in crops, which has been used to create mapping populations for screening. Quantitative trait loci (QTL) for O3 tolerance have been identified in model and crop species, and although none has been cloned to date, transcript profiling experiments have identified candidate genes associated with QTL. Biotechnological strategies for improving O3 tolerance are also being tested, although there is considerable research to be done before O3 -tolerant germplasm is available to growers for most crops. Strategies to improve O3 tolerance in crops have been hampered by the lack of translation of laboratory experiments to the field, and the lack of correlation between visual leaf-level O3 damage and yield loss to O3 stress. Future efforts to screen mapping populations in the field and to identify more promising phenotypes for O3 tolerance are needed.


Normalization of Tumor Vasculature by Oxygen Microbubbles with Ultrasound.
Tumor microenvironment influences the efficacy of anti-cancer therapies. The dysfunctional tumor vasculature limits the efficiency of oxygenation and drug delivery to reduce treatment outcome. A concept of tumor vascular normalization (VN), which inhibits angiogenesis to improve vessel maturity, blood perfusion, and oxygenation, has been demonstrated under the anti-angiogenic therapy. The efficiency of drug delivery and penetration is increased by enhancing perfusion and reducing interstitial fluid pressure during the time window of VN. However, anti-angiogenic agents only induce transient VN and then prune vessels to aggravate tumor hypoxia. To repair tumor vessels without altering vessel density, we proposed to induce tumor VN by local oxygen release via oxygen microbubbles with ultrasound. With tumor perfusion enhancement under ultrasound contrast imaging tracing, the time window of VN was defined as 2-8 days after a single oxygen microbubble treatment. The enhanced tumor oxygenation after oxygen microbubble treatment inhibited hypoxia inducible factor-1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF) pathway to improve the morphology and function of tumor vasculature. The pericyte coverage and Hoechst penetration of tumor vessels increased without any changes to the vessel density. Finally, the intratumoral accumulation of anti-cancer drug doxorubicin could be increased 3-4 folds during tumor VN. These findings demonstrate that regulating tumor oxygenation by oxygen microbubbles could normalize dysfunctional vessels to enhance vascular maturity, blood perfusion, and drug penetration. Furthermore, ultrasound perfusion imaging provides a simple and non-invasive way to detect the VN time window, which increases the feasibility of VN in clinical cancer applications.


Diurnal regulation of sphingolipids in blood.
Key homeostatic functions are regulated in a diurnal manner and a miss-alignment of such rhythms is believed to contribute to the pathophysiology of several diseases. Signaling sphingolipids (SLs) in plasma such as sphingosine 1-phosphate control lymphocytic trafficking, vascular reactivity and platelet activity, physiological functions all of which display a diurnal rhythm themselves. However, the rhythmicity of SL metabolism in plasma and its potential causes have not been sufficiently investigated so far. Therefore, we analyzed blood of mice and healthy adult human subjects by targeted tandem mass-spectrometry at different time points. In order to investigate the influence of the synchronizing hormone melatonin, we compared melatonin proficient C3H/HeN wildtype mice (C3H) with melatonin receptor-1/2 double knockout mice (MT1/2-/-) and melatonin deficient C57BL/6J mice. We found a strong upregulation of plasma S1P with the beginning of the light period in C3H but not in MT1/2-/- or C57BL/6J mice. Accordingly, our study revealed an upregulation of sphingosine 1-phosphate (S1P d18:1) and sphinganine 1-phosphate (S1P d18:0) with the beginning of the light period in humans. Furthermore, plasma S1P d18:1 and S1P d18:0 were inversely correlated with the respective concentrations in platelets, pointing to a possible involvement of platelet SL metabolism. In humans, the diurnal rhythm of SLs was not associated with changes of SL-binding proteins or counts of cellular SL sources. Overall, this study indicates a physiological rhythmicity of plasma and platelet SL metabolism, likely mediated by melatonin, with potentially important implications for physiological diurnal rhythms and the regulation of SL metabolism and its functions.


Accurate Binding of Sodium and Calcium to a POPC Bilayer by Effective Inclusion of Electronic Polarization.
Binding affinities and stoichiometries of Na+ and Ca2+ ions to phospholipid bilayers are of paramount significance in the properties and functionality of cellular membranes. Current estimates of binding affinities and stoichiometries of cations are, however, inconsistent due to limitations in the available experimental and computational methods. In this work, we improve the description of the binding details of Na+ and Ca2+ ions to a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer by implicitly including electronic polarization as a mean field correction, known as the electronic continuum correction (ECC). This is applied by scaling the partial charges of a selected state-of-the-art POPC lipid model for molecular dynamics simulations. Our improved ECC-POPC model reproduces not only the experimentally measured structural parameters for the ion-free membrane, but also the response of lipid headgroup to a strongly bound cationic amphiphile, as well as the binding affinities of Na+ and Ca2+ ions. With our new model, we observe on the one side negligible binding of Na+ ions to POPC bilayer, while on the other side stronger interactions of Ca2+ primarily with phosphate oxygens, which is in agreement with the previous interpretations of the experimental spectroscopic data. The present model results in Ca2+ ions forming complexes with one to three POPC molecules with almost equal probabilities, suggesting more complex binding stoichiometries than those from simple models used to interpret the NMR data previously. The results of this work pave the way to quantitative molecular simulations with realistic electrostatic interactions of complex biochemical systems at cellular membranes.


Medical and dental students' attitudes about the AIDS epidemic.
This study investigated changes, over a one-year period, in medical and dental students' attitudes toward various issues related to contact with AIDS patients. In 1988 and 1989, the authors surveyed second- and third-year medical and dental students at one medical school both before and after they completed a year of required clinical training. The dental students remained consistently more anxious and more restrictive in their attitudes toward treating patients with AIDS than did their medical student counterparts, and became more conservative (fearful) in their attitudes after working with AIDS patients, whereas the medical students became more liberal. Overall, both groups were more concerned about contracting AIDS in their professional lives than in their personal lives. Over one-third of the medical students and two-thirds of the dental students indicated that they did not wish to train in a specialty or hospital with a high percentage of AIDS patients, and a substantial minority of the students in both groups did not feel that they were responsible for treating all patients whom they were qualified to treat. These and related results suggest that AIDS-related anxiety may influence students' career choices and behaviors and the quality of care patients receive. Suggestions for educational strategies to address students' fears--both warranted and unwarranted--and the differences in attitudes between students of different health-care professions are discussed.


Dynamics of relationship between the presence of Coxiella burnetii DNA, antibodies, and intrinsic variables in cow milk and bulk tank milk from Danish dairy cattle.
Milk samples of 12 Danish dairy herds were collected 3 times during an 11-mo period and tested for Coxiella burnetii DNA by real-time PCR, detecting the IS1111 element, and for the presence of antibodies against the bacterium by ELISA. On average, 25% of 1,514 samples were seropositive and 32% were positive for C. burnetii DNA. Among the 485 DNA-positive samples, quantification cycle values ranging from 15.8 to 37.8 were found. Test sensitivity did not increase after DNA extraction from the cream fraction compared with full milk. The relationship between antibody levels and bacterial shedding was investigated among 166 cows from 9 herds. The prevalence levels of C. burnetii DNA and antibodies in the herds were found to be rather stable for 6 of the herds. The test results were highly influenced by results obtained 3 to 7 mo earlier. A significant association between the antibody titer and the DNA shedding level at the same and the preceding visit was found. In addition, a significant association between the antibody titer and the antibody titers 3 to 11 mo earlier was found. A multivariable analysis identified a significant increase in C. burnetii DNA shedding with increasing parity and increasing protein concentration in milk. The antibody levels in bulk tank milk and prevalence levels of C. burnetii DNA and antibodies in individual cow milk samples were correlated. A significant correlation was also found between the quantification cycle values of the cow samples (weighted according to milk yield) and the C. burnetii concentration in bulk tank milk.


Immunogenicity and protective efficacy of recombinant vaccine based on the receptor-binding domain of the Plasmodium vivax Duffy binding protein in Aotus monkeys.
Invasion of human erythrocytes by Plasmodium vivax requires interaction between Duffy binding protein (PvDBP) and the Duffy blood group antigen. The receptor-binding domain of PvDBP lies in a conserved N-terminal, cysteine-rich region, region II (PvRII). PvRII is a valuable malaria subunit vaccine candidate for asexual blood stages. We have evaluated in Aotus monkeys the immunogenicity and protective efficacy of recombinant PvRII formulated in Freund's and Montanide ISA720 adjuvants. Specific antibody titers were determined by an enzyme-linked immunosorbent assay after each of three doses of 50 microg of protein administered by the subcutaneous route. Immunization with PvRII formulated in Freund's adjuvant yielded higher antibody titers than immunization with the Montanide ISA720 formulation and offered partial protection. Although the Montanide ISA720 formulation was immunogenic, it did not provide any protection. Given the immunogenicity and partial protection observed, further studies are needed to optimize the PvRII vaccine formulation with adjuvants suitable for human use.


Pain in dementia.
The ageing revolution is changing the composition of our society with more people becoming very old with higher risks for developing both pain and dementia. Pain is normally signaled by verbal communication, which becomes more and more deteriorated in people with dementia. Thus, these individuals unnecessarily suffer from manageable but unrecognized pain. Pain assessment in patients with dementia is a challenging endeavor, with scientific advancements quickly developing. Pain assessment tools and protocols (mainly observational scales) have been incorporated into national and international guidelines of pain assessment in aged individuals. To effectively assess pain, interdisciplinary collaboration (nurses, physicians, psychologists, computer scientists, and engineers) is essential. Pain management in this vulnerable population is also preferably done in an interdisciplinary setting. Nonpharmacological management programs have been predominantly tested in younger populations without dementia. However, many of them are relatively safe, have proven their efficacy, and therefore deserve a first place in pain management programs. Paracetamol is a relatively safe and effective first-choice analgesic. There are many safety issues regarding nonsteroidal anti-inflammatory drugs, opioids, and adjuvant analgesics in dementia patients. It is therefore recommended to monitor both pain and potential side effects regularly. More research is necessary to provide better guidance for pain management in dementia.


Parents and family members in the era of ART: evidence from Cambodia and Thailand.
Ensuring treatment adherence is critical for the success of antiretroviral therapy (ART) programs in developing countries. Enlisting NGOs or persons living with HIV/AIDS (PLHA) group members as treatment supporters is one common strategy. Less attention is given to family members and especially older-age parents. Yet ART patients often live with other family members who are highly motivated to ensure treatment success. This study examines the role of family members and especially parents in assisting adherence in Cambodia and Thailand among adult ART patients. Most have a living parent and many live with or near a parent. Family members including parents commonly remind patients to take medications, particularly if coresident in the same household. Parents also remind patients to get resupplies and accompany them to appointments. Some contrasts between Cambodia and Thailand emerged. Fewer Cambodian than Thai patients had a living parent. However, among those who did, equal shares lived with parents. Cambodian parents more commonly reminded patients to take medications and get resupplies and accompanied them when doing so. In both countries correct knowledge of ART among parents was associated with the amount of advice from program personnel. The results underscore both the need to more explicitly incorporate close family members, including parents, into efforts to promote adherence and need for PLHA peers and home based care teams to provide them with adequate information, training and resources to increase their effectiveness.


Fatigue of Dental Implants: Facts and Fallacies.
Dental implants experience rare yet problematic mechanical failures such as fracture that are caused, most often, by (time-dependent) metal fatigue. This paper surveys basic evidence about fatigue failure, its identification and the implant's fatigue performance during service. We first discuss the concept of dental implant fatigue, starting with a review of basic concepts related to this failure mechanism. The identification of fatigue failures using scanning electron microscopy follows, to show that this stage is fairly well defined. We reiterate that fatigue failure is related to the implant design and its surface condition, together with the widely varying service conditions. The latter are shown to vary to an extent that precludes devising average or representative conditions. The statistical nature of the fatigue test results is emphasized throughout the survey to illustrate the complexity in evaluating the fatigue behavior of dental implants from a design perspective. Today's fatigue testing of dental implants is limited to ISO 14801 standard requirements, which ensures certification but does not provide any insight for design purposes due to its limited requirements. We introduce and discuss the random spectrum loading procedure as an alternative to evaluate the implant's performance under more realistic conditions. The concept is illustrated by random fatigue testing in 0.9% saline solution.


Secure attachment promotes the development of effortful control in boys.
Although effortful control (EC), a regulatory aspect of temperament, is associated with a wide range of developmental outcomes, knowledge about EC promoters is scarce. This study explored whether secure attachment promoted the development of EC from preschool to school age in a community sample of 903 Norwegian children. EC was measured using the parent-reported Children's Behavior Questionnaire at four (T1) and six (T2) years of age, and attachment was measured using the Manchester Child Attachment Story Task at T1. Previous research has indicated that a child's gender and socioeconomic status are possible covariates of EC; hence, these factors were included in the analyses. Despite considerable rank-order stability in EC, secure attachment contributed to an increase in EC. Furthermore, gender moderated the effect of attachment: secure attachment promoted EC in boys only. These findings emphasize preschool boys' need for emotional security to facilitate effortful capacities in their transition to school.


Local structural changes caused by peptidyl-prolyl cis/trans isomerization in the native state of proteins.
Peptidyl-prolyl cis/trans isomerization, observed in the native state of an increasing number of proteins, is of considerable biological significance. The first evidence for an asymmetric transmission along the polypeptide chain of the structural effects of prolyl isomerization is now derived from the statistics of the C(alpha)/C(alpha)-atom distance distributions in the crystal structures of 848 non-homologous proteins. More detailed information on how isomerization affects segments adjacent to proline is obtained from crystal structures of proteins, that are more than 95% homologous, and that exhibit two different states of isomerization at a particular prolyl bond. The resulting 64 cases, which represent 3.8% of the database used, form pairs of coordinates which were analyzed for the existence of isomer-specific intramolecular nonbonded C(alpha)/C(alpha)-atom distances around the critical proline, and for the positional preferences for particular amino acids in the isomeric sequence segment. The probability that a native protein exhibits both prolyl isomers in the crystalline state increases in particular with a Pro at the third position N-terminal to the isomeric bond (-3 position), and with Ser, Gly and Asp at the position preceding the isomeric bond (-1 position). Structural alignment of matched pairs of isomeric proteins generates three classes with respect to position-specific distribution of C(alpha)-atom displacements around an isomeric proline imide bond. In the majority of cases the distribution of these intermolecular isomer-specific C(alpha)-atom distances shows a symmetric behavior for the N-terminal and C-terminal segment flanking the proline residue, and the magnitude did not exceed 1.3+/-0.6 A including the C(alpha) atoms in proximity to the prolyl bond. However, in the remaining 12 protein pairs the structural changes are unidirectional relative to the isomerizing bond whereby the magnitude of the isomer-specific effect exceeds 3.0+/-2.0 A even at positions remote to proline. Interestingly, the magnitude of the intramolecular isomer-specific C(alpha) atom displacements reveals a lever-arm amplification of the isomerization-mediated structural changes in a protein backbone. The observed backbone effects provide a structural basis for isomer-specific reactions of proline-containing polypeptides, and thus may play a role in biological recognition and regulation.


Blood transfusion and risk of metastatic disease or recurrence in patients undergoing immediate TRAM flap breast reconstruction: a clinical study and meta-analysis.
The transfusion of blood products has a known immunomodulatory effect that may affect cancer recurrence. The present study examined whether blood transfusion is an independent risk factor for recurrence or development of metastatic disease among patients undergoing immediate breast reconstruction with a transverse rectus abdominis musculocutaneous (TRAM) flap. Records of 103 patients who underwent mastectomy and immediate reconstruction with a TRAM flap between 1991 and 2001 were reviewed. A logistic regression analysis was used to identify independent risk factors for metastasis or recurrence. For the meta-analysis, all English-language studies regarding blood transfusion and breast cancer recurrence were reviewed, and 2 x 2 contingency tables were constructed from which a summary relative risk was calculated. There were 57 free and 35 pedicle TRAM flaps. Forty-nine patients (48 percent) received perioperative transfusion of nonautologous blood. Twenty patients (19 percent) experienced metastatic disease or local recurrence. Follow-up ranged from 4 to 14 years (mean, 6.7 years). There was a higher observed rate of adverse outcome in patients who received transfusion, but this was not statistically significant (p = 0.90). Of the 11 articles identified by the meta-analysis, eight used a regression analysis controlling for the effect of stage and nodal status. The summary relative risk in these studies was 1.03 (95% CI, 0.90 to 1.26). Perioperative blood transfusion does not seem to be an independent risk factor for metastasis or cancer recurrence in patients undergoing TRAM flap reconstruction. The observed correlation in this and prior studies may be due to the effect of other, more significant factors, such as tumor stage and nodal status.


The impact of resident duty hour reform on hospital readmission rates among Medicare beneficiaries.
A key goal of resident duty hour reform by the Accreditation Council for Graduate Medical Education (ACGME) in 2003 was to improve patient outcomes. To assess whether the reform led to a change in readmission rates. Observational study using multiple time series analysis with hospital discharge data from July 1, 2000 to June 30, 2005. Fixed effects logistic regression was used to examine the change in the odds of readmission in more versus less teaching-intensive hospitals before and after duty hour reform. All unique Medicare patients (n = 8,282,802) admitted to acute-care nonfederal hospitals with principal diagnoses of acute myocardial infarction, congestive heart failure, gastrointestinal bleeding, or stroke (combined medical group), or a DRG classification of general, orthopedic, or vascular surgery (combined surgical group). Primary outcome was 30-day all-cause readmission. Secondary outcomes were (1) readmission or death within 30 days of discharge, and (2) readmission, death during the index admission, or death within 30 days of discharge. For the combined medical group, there was no evidence of a change in readmission rates in more versus less teaching-intensive hospitals [OR = 0.99 (95% CI 0.94, 1.03) in post-reform year 1 and OR = 0.99 (95% CI 0.95, 1.04) in post-reform year 2]. There was also no evidence of relative changes in readmission rates for the combined surgical group: OR = 1.03 (95% CI 0.98, 1.08) for post-reform year 1 and OR = 1.02 (95% CI 0.98, 1.07) for post-reform year 2. Findings for the secondary outcomes combining readmission and death were similar. Among Medicare beneficiaries, there were no changes in hospital readmission rates associated with resident duty hour reform.


Nuclear translocation and functions of growth factor receptors.
Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?


Docetaxel and mitoxantrone before radical prostatectomy in men with high-risk prostate cancer: 10-year follow-up and immune correlates.
The aims of this study were to report the clinical outcomes in a cohort of men with high-risk prostate cancer treated with neoadjuvant docetaxel and mitoxantrone 10 years after treatment, identify pretreatment clinical parameters that may be predictors of recurrence, and describe tumor-infiltrating leukocytes present in radical prostatectomy specimens. We conducted a phase I/II study of neoadjuvant docetaxel and mitoxantrone before radical prostatectomy in high-risk localized prostate cancer to determine the feasibility of this combination and predictors of prostate cancer recurrence after cytotoxic chemotherapy. After 10 years of follow-up, 34 (63%) of 54 participants experience a recurrence. In univariate analysis, prostate-specific antigen (PSA) density (P=0.01), pathological stage (P=0.03), lymph node status (P<0.0001), seminal vesicle invasion (P=0.003), and tissue vascular endothelial growth factor (VEGF) expression (P=0.016) were significantly associated with recurrence. In multivariate analysis, only lymph node status, PSA density, and VEGF expression were significant predictors of disease recurrence. We used a tissue microarray for the first 50 participants to characterize the tumor-infiltrating lymphocytes and evaluate them for association with recurrence. We measured CD3, CD4, CD8, FoxP3, CD20, CD15, CD68, and CD163 by immunohistochemistry in both tumor and normal prostate specimens, but did not find an association between immunophenotype and recurrence. There was a significantly different density of CD68 and CD163 cells between normal and tumor tissue. Lymph node status, PSA density, and tissue VEGF expression predict recurrence after chemotherapy for high-risk prostate cancer. Additional studies are needed to determine the potential benefit of chemotherapy in the neoadjuvant setting.


Next generation sequencing and de novo transcriptome analysis of Costus pictus D. Don, a non-model plant with potent anti-diabetic properties.
Phyto-remedies for diabetic control are popular among patients with Type II Diabetes mellitus (DM), in addition to other diabetic control measures. A number of plant species are known to possess diabetic control properties. Costus pictus D. Don is popularly known as "Insulin Plant" in Southern India whose leaves have been reported to increase insulin pools in blood plasma. Next Generation Sequencing is employed as a powerful tool for identifying molecular signatures in the transcriptome related to physiological functions of plant tissues. We sequenced the leaf transcriptome of C. pictus using Illumina reversible dye terminator sequencing technology and used combination of bioinformatics tools for identifying transcripts related to anti-diabetic properties of C. pictus. A total of 55,006 transcripts were identified, of which 69.15% transcripts could be annotated. We identified transcripts related to pathways of bixin biosynthesis and geraniol and geranial biosynthesis as major transcripts from the class of isoprenoid secondary metabolites and validated the presence of putative norbixin methyltransferase, a precursor of Bixin. The transcripts encoding these terpenoids are known to be Peroxisome Proliferator-Activated Receptor (PPAR) agonists and anti-glycation agents. Sequential extraction and High Performance Liquid Chromatography (HPLC) confirmed the presence of bixin in C. pictus methanolic extracts. Another significant transcript identified in relation to anti-diabetic, anti-obesity and immuno-modulation is of Abscisic Acid biosynthetic pathway. We also report many other transcripts for the biosynthesis of antitumor, anti-oxidant and antimicrobial metabolites of C. pictus leaves. Solid molecular signatures (transcripts related to bixin, abscisic acid, and geranial and geraniol biosynthesis) for the anti-diabetic properties of C. pictus leaves and vital clues related to the other phytochemical functions like antitumor, anti-oxidant, immuno-modulatory, anti-microbial and anti-malarial properties through the secondary metabolite pathway annotations are reported. The data provided will be of immense help to researchers working in the treatment of DM using herbal therapies.


Introduction to opportunities and pitfalls in functional mass spectrometry based proteomics.
With the advent of mass spectrometry based proteomics, the identification of thousands of proteins has become commonplace in biology nowadays. Increasingly, efforts have also been invested toward the detection and localization of posttranslational modifications. It is furthermore common practice to quantify the identified entities, a task supported by a panel of different methods. Finally, the results can also be enriched with functional knowledge gained on the proteins, detecting for instance differentially expressed gene ontology terms or biological pathways. In this study, we review the resources, methods and tools available for the researcher to achieve such a quantitative functional analysis. These include statistics for the post-processing of identification and quantification results, online resources and public repositories. With a focus on free but user-friendly software, preferably also open-source, we provide a list of tools designed to help the researcher manage the vast amount of data generated. We also indicate where such applications currently remain lacking. Moreover, we stress the eventual pitfalls of every step of such studies. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.


Cold-induced increase in brown fat thyroxine 5'-monodeiodinase is attenuated in Zucker obese rat.
In this study we examined the possibility that the reduced brown adipose tissue (BAT) thermogenesis in the Zucker obese rat may result from a limited capacity for enzymic conversion of thyroxine (T4) to triiodothyronine (T3) in BAT. A total of 34 lean and obese rats, approximately 4 mo old were divided into three treatment groups: group 1 (5 lean and 6 obese) was fed Purina rat chow for 21 days, and group 2 (5 lean and 6 obese) was fed a cafeteria diet for 21 days, and group 3 (6 lean and 6 obese) was fed Purina rat chow and maintained in the cold (8 +/- 1 degrees C) for 7 days. The lean and obese rats in all three groups of animals were matched closely for age and respective body weight. Activity of T4 5'-deiodinase was determined as the rate of T3 production from added T4 under controlled in vitro conditions. Serum T4 and T3 were determined by radioimmunoassay. The rate of T4-to-T3 conversion in BAT was similar in the lean and obese rats maintained at room temperature, whether fed rat chow or a cafeteria diet (approximately 40-50 pmol T3/scapular BAT depot per h). However, expressed per scapular BAT depot, lean rats exposed to cold displayed about a fivefold increase in BAT T3 production (P less than 0.0001), whereas only a small increase was observed in the cold-exposed obese rats. Serum T3 levels tended to be reduced in the Zucker obese rats. Our data indicate a reduced capacity for T3 production in Zucker rat BAT exposed to cold.(ABSTRACT TRUNCATED AT 250 WORDS)


An updated investigation of the multidimensional structure of language lateralization in left- and right-handed adults: a test-retest functional transcranial Doppler sonography study with six language tasks.
A previous study we reported in this journal suggested that left and right-handers may differ in their patterns of lateralization for different language tasks (Woodhead et al. 2019 R. Soc. Open Sci. 6, 181801. (doi:10.1098/rsos.181801)). However, it had too few left-handers (N = 7) to reach firm conclusions. For this update paper, further participants were added to the sample to create separate groups of left- (N = 31) and right-handers (N = 43). Two hypotheses were tested: (1) that lateralization would be weaker at the group level in left-than right-handers; and (2) that left-handers would show weaker covariance in lateralization between tasks, supporting a two-factor model. All participants performed the same protocol as in our previous paper: lateralization was measured using functional transcranial Doppler sonography during six different language tasks, on two separate testing sessions. The results supported hypothesis 1, with significant differences in laterality between groups for four out of six tasks. For hypothesis 2, structural equation modelling showed that there was stronger evidence for a two-factor model in left than right-handers; furthermore, examination of the factor loadings suggested that the pattern of laterality across tasks may also differ between handedness groups. These results expand on what is known about the differences in laterality between left- and right-handers.


Methylglyoxal and D-lactate in cisplatin-induced acute kidney injury: Investigation of the potential mechanism via fluorogenic derivatization liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) proteomic analysis.
Nephrotoxicity severely limits the chemotherapeutic efficacy of cisplatin (CDDP). Oxidative stress is associated with CDDP-induced acute kidney injury (AKI). Methylglyoxal (MG) forms advanced glycation end products that elevate oxidative stress. We aimed to explore the role of MG and its metabolite D-lactate and identify the proteins involved in CDDP-induced AKI. Six-week-old female BALB/c mice were intraperitoneally administered CDDP (5 mg/kg/day) for 3 or 5 days. Blood urea nitrogen (42.6 ± 7.4 vs. 18.3 ± 2.5; p < 0.05) and urinary N-acetyl-β-D-glucosaminide (NAG; 4.89 ± 0.61 vs. 2.43 ± 0.31 U/L; p < 0.05) were significantly elevated in the CDDP 5-day group compared to control mice. Histological analysis confirmed AKI was successfully induced. Confocal microscopy revealed TNF-α was significantly increased in the CDDP 5-day group. Fluorogenic derivatized liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) showed the kidney MG (36.25 ± 1.68 vs. 18.95 ± 2.24 mg/g protein, p < 0.05) and D-lactate (1.78 ± 0.29 vs. 1.12 ± 0.06 mol/g protein, p < 0.05) contents were significantly higher in the CDDP 5-day group than control group. FD-LC-MS/MS proteomics identified 33 and nine altered peaks in the CDDP 3-day group and CDDP 5-day group (vs. control group); of the 35 proteins identified using the MOSCOT database, 11 were antioxidant-related. Western blotting confirmed that superoxide dismutase 1 (SOD-1) and parkinson disease protein 7 (DJ-1) are upregulated and may participate with MG in CDDP-induced AKI. This study demonstrates TNF-α, MG, SOD-1 and DJ-1 play crucial roles in CDDP-induced AKI.


Influence of Dietary Sources of Melatonin on Sleep Quality: A Review.
Sleep is an essential biological phenomenon, being a physiological and behavioral process necessary for quality of life. Melatonin is a circadian hormone produced at night by the pineal gland, regulated by the light/dark cycle, under the control of the suprachiasmatic nucleus. Melatonin is an indoleamine, synthesized from the essential amino acid tryptophan via serotonin. Melatonin is also found in plants, where it helps fight oxidative stress. To present a systematic review on the ability of food sources of melatonin to promote healthy sleep. A literature search was performed on the PubMed, Scopus, and ScienceDirect databases, including only randomized, placebo-controlled trials published in English between 2005 and 2019. The methodological quality of the trials was assessed by the Jadad scale. Of the 25 eligible articles, eight met the inclusion criteria. They addressed the intake of milk or cherry juice in children, adults, and elderly subjects and evaluated sleep quality by questionnaires, sleep diary, actigraphy, or polysomnography. The analysis of the studies presented limitations, including lack of homogeneity of treatment dosage and duration. Nonetheless, the results indicated that the consumption of milk and sour cherries, sources of melatonin, may improve sleep quality in humans. These results pointed out to the potential suitability of food sources of melatonin as adjuvants in the prevention and treatment of sleep disorders. Further studies are necessary to better ascertain the aspects relevant to their use.


Optical imaging of ovarian cancer using a matrix metalloproteinase-3-sensitive near-infrared fluorescent probe.
Epithelial ovarian cancer (EOC) is the seventh most common cancer among women worldwide. The 5-year survival rate for women with EOC is only 30%-50%, which is largely due to the typically late diagnosis of this condition. EOC is difficult to detect in its early stage because of its asymptomatic nature. Recently, near-infrared fluorescent (NIRF) imaging has been developed as a potential tool for detecting EOC at the molecular level. In this study, a NIRF-sensitive probe was designed to detect matrix metalloproteinase (MMP) activity in ovarian cancer cells. A cyanine fluorochrome was conjugated to the amino terminus of a peptide substrate with enzymatic specificity for MMP-3. To analyze the novel MMP-3 probe, an in vivo EOC model was established by subcutaneously implanting SKOV3 cells, a serous-type EOC cell line, in mice. This novel MMP-3-sensitive probe specifically reacted with only the active MMP-3 enzyme, resulting in a significantly enhanced NIRF emission intensity. Histological analysis demonstrated that MMP-3 expression and activity were enhanced in the stromal cells surrounding the ovarian cancer cells. These studies establish a molecular imaging reporter for diagnosing early-stage EOC. Additional studies are required to confirm the early-stage activity of MMP-3 in EOC and its diagnostic and prognostic significance.


International benchmarking of specialty hospitals. A series of case studies on comprehensive cancer centres.
Benchmarking is one of the methods used in business that is applied to hospitals to improve the management of their operations. International comparison between hospitals can explain performance differences. As there is a trend towards specialization of hospitals, this study examines the benchmarking process and the success factors of benchmarking in international specialized cancer centres. Three independent international benchmarking studies on operations management in cancer centres were conducted. The first study included three comprehensive cancer centres (CCC), three chemotherapy day units (CDU) were involved in the second study and four radiotherapy departments were included in the final study. Per multiple case study a research protocol was used to structure the benchmarking process. After reviewing the multiple case studies, the resulting description was used to study the research objectives. We adapted and evaluated existing benchmarking processes through formalizing stakeholder involvement and verifying the comparability of the partners. We also devised a framework to structure the indicators to produce a coherent indicator set and better improvement suggestions. Evaluating the feasibility of benchmarking as a tool to improve hospital processes led to mixed results. Case study 1 resulted in general recommendations for the organizations involved. In case study 2, the combination of benchmarking and lean management led in one CDU to a 24% increase in bed utilization and a 12% increase in productivity. Three radiotherapy departments of case study 3, were considering implementing the recommendations.Additionally, success factors, such as a well-defined and small project scope, partner selection based on clear criteria, stakeholder involvement, simple and well-structured indicators, analysis of both the process and its results and, adapt the identified better working methods to the own setting, were found. The improved benchmarking process and the success factors can produce relevant input to improve the operations management of specialty hospitals.


Artificial limb representation in amputees.
The human brain contains multiple hand-selective areas, in both the sensorimotor and visual systems. Could our brain repurpose neural resources, originally developed for supporting hand function, to represent and control artificial limbs? We studied individuals with congenital or acquired hand-loss (hereafter one-handers) using functional MRI. We show that the more one-handers use an artificial limb (prosthesis) in their everyday life, the stronger visual hand-selective areas in the lateral occipitotemporal cortex respond to prosthesis images. This was found even when one-handers were presented with images of active prostheses that share the functionality of the hand but not necessarily its visual features (e.g. a 'hook' prosthesis). Further, we show that daily prosthesis usage determines large-scale inter-network communication across hand-selective areas. This was demonstrated by increased resting state functional connectivity between visual and sensorimotor hand-selective areas, proportional to the intensiveness of everyday prosthesis usage. Further analysis revealed a 3-fold coupling between prosthesis activity, visuomotor connectivity and usage, suggesting a possible role for the motor system in shaping use-dependent representation in visual hand-selective areas, and/or vice versa. Moreover, able-bodied control participants who routinely observe prosthesis usage (albeit less intensively than the prosthesis users) showed significantly weaker associations between degree of prosthesis observation and visual cortex activity or connectivity. Together, our findings suggest that altered daily motor behaviour facilitates prosthesis-related visual processing and shapes communication across hand-selective areas. This neurophysiological substrate for prosthesis embodiment may inspire rehabilitation approaches to improve usage of existing substitutionary devices and aid implementation of future assistive and augmentative technologies.


Prediction of protein secondary structures with a novel kernel density estimation based classifier.
Though prediction of protein secondary structures has been an active research issue in bioinformatics for quite a few years and many approaches have been proposed, a new challenge emerges as the sizes of contemporary protein structure databases continue to grow rapidly. The new challenge concerns how we can effectively exploit all the information implicitly deposited in the protein structure databases and deliver ever-improving prediction accuracy as the databases expand rapidly. The new challenge is addressed in this article by proposing a predictor designed with a novel kernel density estimation algorithm. One main distinctive feature of the kernel density estimation based approach is that the average execution time taken by the training process is in the order of O(nlogn), where n is the number of instances in the training dataset. In the experiments reported in this article, the proposed predictor delivered an average Q3 (three-state prediction accuracy) score of 80.3% and an average SOV (segment overlap) score of 76.9% for a set of 27 benchmark protein chains extracted from the EVA server that are longer than 100 residues. The experimental results reported in this article reveal that we can continue to achieve higher prediction accuracy of protein secondary structures by effectively exploiting the structural information deposited in fast-growing protein structure databases. In this respect, the kernel density estimation based approach enjoys a distinctive advantage with its low time complexity for carrying out the training process.


Stress, drink, leave: An examination of gender-specific risk factors for mental health problems and attrition among licensed attorneys.
Rates of mental illness and heavy alcohol use are exceedingly high in the legal profession, while attrition among women has also been a longstanding problem. Work overcommitment, work-family conflict, permissiveness toward alcohol in the workplace, and the likelihood of promotion are all implicated but have yet to be systematically investigated. Data were collected from 2,863 lawyers randomly sampled from the California Lawyers Association and D.C. Bar to address this knowledge gap. Findings indicated that the prevalence and severity of depression, anxiety, stress, and risky/hazardous drinking were significantly higher among women. Further, one-quarter of all women contemplated leaving the profession due to mental health concerns, compared to 17% of men. Logistic models were conducted to identify workplace factors predictive of stress, risky drinking, and contemplating leaving the profession. Overcommitment and permissiveness toward alcohol at work were associated with the highest likelihood of stress and risky drinking (relative to all other predictors) for both men and women. However, women and men differed with respect to predictors of leaving the profession due to stress or mental health. For women, work-family conflict was associated with the highest likelihood of leaving, while overcommitment was the number one predictor of leaving for men. Mental health and gender disparities are significant problems in the legal profession, clearly requiring considerable and sustained attention.


Folate intake, post-folic acid grain fortification, and pancreatic cancer risk in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial.
Folate plays a critical role in DNA methylation, synthesis, and repair. Several epidemiologic studies suggest that higher folate intake is associated with decreased pancreatic cancer risk. We investigated the association between dietary folate intake and pancreatic cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) cohort. Dietary data were collected with the use of a self-administered food-frequency questionnaire (1998-2005). Among the 51,988 male and 57,187 female participants, aged 55-74 y at enrollment, with complete dietary and multivitamin information, 162 men and 104 women developed pancreatic cancer during follow-up (January 1998 to December 2006; median: 6.5 y). We used Cox proportional hazards regression with age as the time metric to calculate hazard ratios (HRs) and 95% CIs. The highest compared with the lowest quartile of food folate was associated with a significantly decreased pancreatic cancer risk among women (> or = 253.3 compared with < or = 179.1 microg/d; HR = 0.47; 95% CI: 0.23, 0.94; P for trend: 0.09) but not among men (> or = 229.6 compared with < or = 158.0 microg/d; HR = 1.20; 95% CI: 0.70, 2.04; P for trend: 0.67; P for interaction by sex: 0.03). There was also a significant inverse trend in risk of pancreatic cancer across increasing quartiles of total folate in women (P for trend: 0.04) but not in men (P for trend: 0.65). Folic acid supplements were not associated with pancreatic cancer. These findings support an association between higher food and total folate intakes and decreased risk of pancreatic cancer in women but not in men.


Hematotoxicity of the chinese herbal medicine Tripterygium wilfordii hook f in CD34-positive human bone marrow cells.
T2, a chloroform/methanol extract of the herb Tripterygium wilfordii Hook f, has been used in China for the treatment of autoimmune and inflammatory diseases for many years. Recent experimental evidence has confirmed that T2 has potent anti-inflammatory and immunosuppressive activity, and a United States Food and Drug Administration-approved clinical trial is currently exploring the efficacy of T2 in the treatment of rheumatoid arthritis. Despite the potential therapeutic benefits of T2, there is ample documentation that T2 is toxic, targeting, among other things, the hematopoietic system, and its use has resulted in cases of leukopenia, thrombocytopenia, and aplastic anemia. This investigation was undertaken to characterize the in vitro effects of T2 on primary human CD34-positive (CD34+) bone marrow cells. Our results demonstrate that T2 has a potent inhibitory effect on the clonogenic response of human bone marrow cells to exogenously added hematopoietic growth factors. The inhibition of colony formation by T2 is not the result of direct cytotoxicity or increased apoptosis and indicates a functional suppression of hematopoiesis. Additional experiments demonstrate that T2 also alters transcriptional regulation in bone marrow cells by inhibiting nuclear factor-kappaB. This transcription factor is found in CD34+ bone marrow cells and has been recently shown to be a requirement for colony formation. These results demonstrate that therapeutic concentrations of T2 exert a significant hematotoxic effect by inhibiting growth factor response in CD34+ bone marrow cells and suggest that inhibition of nuclear factor-kappaB may play a role in the blood dyscrasias encountered with the use of this drug.


Solid-State C-S Coupling in Nickel Organochalcogenide Frameworks as a Route to Hierarchical Structure Transfer to Binary Nanomaterials.
In this work, the transfer of the flexible and easily tunable hierarchical structure of nickel organochalcogenides to different binary Ni-based nanomaterials via selective coupling of organic units was developed. We suggested the use of substituted aryl groups in organosulfur ligands (SAr) as traceless structure-inducing units to prepare nanostructured materials. At the first step, it was shown that the slight variation of the type of SAr units and synthetic procedures allowed us to obtain nickel thiolates [Ni(SAr)2]n with diverse morphologies after a self-assembly process in solution. This feature opened the way for the synthesis of different nanomaterials from a single type of precursor using the phenomenon of direct transfer of morphology. This study revealed that various nickel thiolates undergo selective C-S coupling under high-temperature conditions with the formation of highly demanding nanostructured NiS particles and corresponding diaryl sulfides. The in situ oxidation of the formed nickel sulfide in the case of reaction in an air atmosphere provided another type of valuable nanomaterial, nickel oxide. The high selectivity of the transformation allowed the preservation of the initial organochalcogenide morphologies in the resulting products.


Identification of Culturable Oligotrophic Bacteria within Naturally Occurring Bacterioplankton Communities of the Ligurian Sea by 16S rRNA Sequencing and Probing.
> Abstract Typical marine bacteria (i.e., obligately oligotrophic) that were numerically dominant members of naturally occurring marine communities were identified by cloning and sequencing the amplified 16S rRNA genes obtained from dilution cultures of the original samples. The data reported here refer to two different habitats of a marine pelagic environment (28 miles offshore, in the northwestern Mediterranean Sea). The samples were taken from the water column at two representative layers, i.e., the 30-m depth, corresponding to the chlorophyll maximum layer, and the 1800-m depth, representative of a deep, oligotrophic environment. Three major lineages were found in the 16S rDNA clone libraries prepared from the two samples, two of which could be assigned to the Vibrio and the Rhodobacter groups. The third lineage was a distant relative of the genus Flavobacterium, but it was not closely related to any marine isolate. Six oligonucleotide probes, either complementary to the conserved sequence domains or selectively hybridizing to the clone sequences, were designed for use as hybridization group-specific and strain-specific probes. A single-mismatch discrimination between certain probes and nontarget sequences was demonstrated by detecting the probes' specificity at different hybridization and washing conditions. The screening of the clone libraries with the obtained probes revealed that neither the 30-m sample higher dilution nor the 1800-m one were pure cultures. While some representatives of the Vibrio group were found in both the surface and the deep sample, the members of the Flavobacterium and Rhodobacter lineages were detected only in the deep and the euphotic layers, respectively. We suggest an approach for analyzing autochthonous marine bacteria able to grow in unamended seawater.


MEK Inhibition Induces Canonical WNT Signaling through YAP in KRAS Mutated HCT-15 Cells, and a Cancer Preventive FOXO3/FOXM1 Ratio in Combination with TNKS Inhibition.
The majority of colorectal cancers are induced by subsequent mutations in APC and KRAS genes leading to aberrant activation of both canonical WNT and RAS signaling. However, due to induction of feedback rescue mechanisms some cancers do not respond well to targeted inhibitor treatments. In this study we show that the APC and KRAS mutant human colorectal cancer cell line HCT-15 induces canonical WNT signaling through YAP in a MEK dependent mechanism. This inductive loop is disrupted with combined tankyrase (TNKS) and MEK inhibition. RNA sequencing analysis suggests that combined TNKS/MEK inhibition induces metabolic stress responses in HCT-15 cells promoting a positive FOXO3/FOXM1 ratio to reduce antioxidative and cryoprotective systems.


Aberrant brain responses to emotionally valent words is normalised after cognitive behavioural therapy in female depressed adolescents.
Depression in adolescence is debilitating with high recurrence in adulthood, yet its pathophysiological mechanism remains enigmatic. To examine the interaction between emotion, cognition and treatment, functional brain responses to sad and happy distractors in an affective go/no-go task were explored before and after Cognitive Behavioural Therapy (CBT) in depressed female adolescents, and healthy participants. Eighty-two Depressed and 24 healthy female adolescents, aged 12-17 years, performed a functional magnetic resonance imaging (fMRI) affective go/no-go task at baseline. Participants were instructed to withhold their responses upon seeing happy or sad words. Among these participants, 13 patients had CBT over approximately 30 weeks. These participants and 20 matched controls then repeated the task. At baseline, increased activation in response to happy relative to neutral distractors was observed in the orbitofrontal cortex in depressed patients which was normalised after CBT. No significant group differences were found behaviourally or in brain activation in response to sad distractors. Improvements in symptoms (mean: 9.31, 95% CI: 5.35-13.27) were related at trend-level to activation changes in orbitofrontal cortex. In the follow-up section, a limited number of post-CBT patients were recruited. To our knowledge, this is the first fMRI study addressing the effect of CBT in adolescent depression. Although a bias toward negative information is widely accepted as a hallmark of depression, aberrant brain hyperactivity to positive distractors was found and normalised after CBT. Research, assessment and treatment focused on positive stimuli could be a future consideration. Moreover, a pathophysiological mechanism distinct from adult depression may be suggested and awaits further exploration.


Genetic variation of HIV type 1 in four World Health Organization-sponsored vaccine evaluation sites: generation of functional envelope (glycoprotein 160) clones representative of sequence subtypes A, B, C, and E. WHO Network for HIV Isolation and Characterization.
As part of the WHO Network for HIV Isolation and Characterization, we PCR amplified, cloned, and sequenced gp120 and gp160 genes from 12 HIV-1 isolates collected in four WHO-sponsored vaccine evaluation sites (Brazil, Rwanda, Thailand, Uganda). Envelope clones were derived from PBMC-grown isolates obtained from asymptomatic individuals within 2 years of seroconversion. Analysis of their deduced amino acid sequences identified all but one to contain an uninterrupted open reading frame. Transient expression and biological characterization of selected gp160 constructs identified six clones to encode full length and functional envelope glycoproteins. Phylogenetic analysis of their nucleotide sequences revealed that they represent HIV-1 subtypes A, B, C, and E. Since current knowledge of HIV-1 envelope immunobiology is almost exclusively derived from subtype B viruses, these reagents should facilitate future envelope structure, function and antigenicity studies on a broader spectrum of viruses. This should assist in the design and evaluation of effective vaccines against HIV-1.


Appetite and gut hormone responses to moderate-intensity continuous exercise versus high-intensity interval exercise, in normoxic and hypoxic conditions.
This study investigated the effects of continuous moderate-intensity exercise (MIE) and high-intensity interval exercise (HIIE) in combination with short exposure to hypoxia on appetite and plasma concentrations of acylated ghrelin, peptide YY (PYY), and glucagon-like peptide-1 (GLP-1). Twelve healthy males completed four, 2.6 h trials in a random order: (1) MIE-normoxia, (2) MIE-hypoxia, (3) HIIE-normoxia, and (4) HIIE-hypoxia. Exercise took place in an environmental chamber. During MIE, participants ran for 50 min at 70% of altitude-specific maximal oxygen uptake (V˙O2max) and during HIIE performed 6 × 3 min running at 90% V˙O2max interspersed with 6 × 3 min active recovery at 50% V˙O2max with a 7 min warm-up and cool-down at 70% V˙O2max (50 min total). In hypoxic trials, exercise was performed at a simulated altitude of 2980 m (14.5% O2). Exercise was completed after a standardised breakfast. A second meal standardised to 30% of participants' daily energy requirements was provided 45 min after exercise. Appetite was suppressed more in hypoxia than normoxia during exercise, post-exercise, and for the full 2.6 h trial period (linear mixed modelling, p <0.05). Plasma acylated ghrelin concentrations were lower in hypoxia than normoxia post-exercise and for the full 2.6 h trial period (p <0.05). PYY concentrations were higher in HIIE than MIE under hypoxic conditions during exercise (p = 0.042). No differences in GLP-1 were observed between conditions (p > 0.05). These findings demonstrate that short exposure to hypoxia causes suppressions in appetite and plasma acylated ghrelin concentrations. Furthermore, appetite responses to exercise do not appear to be influenced by exercise modality.


Free Radical-Scavenging, Anti-Inflammatory/Anti-Fibrotic and Hepatoprotective Actions of Taurine and Silymarin against CCl4 Induced Rat Liver Damage.
The present study aims to investigate the hepatoprotective effect of taurine (TAU) alone or in combination with silymarin (SIL) on CCl4-induced liver damage. Twenty five male rats were randomized into 5 groups: normal control (vehicle treated), toxin control (CCl4 treated), CCl4+TAU, CCl4+SIL and CCl4+TAU+SIL. CCl4 provoked significant increases in the levels of hepatic TBARS, NO and NOS compared to control group, but the levels of endogenous antioxidants such as SOD, GPx, GR, GST and GSH were significantly decreased. Serum pro-inflammatory and fibrogenic cytokines including TNF-α, TGF-β1, IL-6, leptin and resistin were increased while the anti-inflammatory (adiponectin) cytokine was decreased in all treated rats. Our results also showed that CCl4 induced an increase in liver injury parameters like serum ALT, AST, ALP, GGT and bilirubin. In addition, a significant increase in liver tissue hydroxyproline (a major component of collagen) was detected in rats exposed to CCl4. Moreover, the concentrations of serum TG, TC, HDL-C, LDL-C, VLDL-C and FFA were significantly increased by CCl4. Both TAU and SIL (i.e., antioxidants) post-treatments were effectively able to relieve most of the above mentioned imbalances. However, the combination therapy was more effective than single applications in reducing TBARS levels, NO production, hydroxyproline content in fibrotic liver and the activity of serum GGT. Combined treatment (but not TAU- or SIL-alone) was also able to effectively prevent CCl4-induced decrease in adiponectin serum levels. Of note, the combined post-treatment with TAU+SIL (but not monotherapy) normalized serum FFA in CCl4-treated rats. The biochemical results were confirmed by histological and ultrastructural changes as compared to CCl4-poisoned rats. Therefore, on the basis of our work, TAU may be used in combination with SIL as an additional adjunct therapy to cure liver diseases such as fibrosis, cirrhosis and viral hepatitis.