The small subunit is required for functional interaction of DNA polymerase delta with the proliferating cell nuclear antigen.
DNA polymerase delta is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA polymerase delta (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA polymerase delta, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA polymerase delta in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA polymerase delta results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA polymerase delta with PCNA and for highly processive DNA synthesis.


6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death.
Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44+CD24-/low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment.


Antifungal activity in thrips soldiers suggests a dual role for this caste.
The social insect soldier is perhaps the most widely known caste, because it often exhibits spectacular weapons, such as highly enlarged jaws or reinforced appendages, which are used to defend the colony against enemies ranging in size from wasps to anteaters. We examined the function of the enlarged forelimbs of soldiers (both male and female) of the eusocial, gall-inhabiting insect Kladothrips intermedius, and discovered that they have little impact on their ability to repel the specialized invading thrips Koptothrips species. While the efficacy of the enlarged forelimb appears equivocal, we show that soldiers secrete strong antifungal compounds capable of controlling the specialized insect fungal pathogen, Cordyceps bassiana. Our data suggest that these thrips soldiers have evolved in response to selection by both macro- and micro-organisms. While it is unknown whether specialized fungal pathogens have been major selective agents in the evolution of the soldier caste in general, they were probably present when sociality first evolved and may have been the primordial enemies of social insects.


Phenotypes and tolerances in the design space of biochemical systems.
One of the major unsolved problems of modern biology is deep understanding of the complex relationship between the information encoded in the genome of an organism and the phenotypic properties manifested by that organism. Fundamental advances must be made before we can begin to approach the goal of predicting the phenotypic consequences of a given mutation or an organism's response to a novel environmental challenge. Although this problem is often portrayed as if the task were to find a more or less direct link between genotypic and phenotypic levels, on closer examination the relationship is far more layered and complex. Although there are some intuitive notions of what is meant by phenotype at the level of the organism, it is far from clear what this term means at the biochemical level. We have described design principles that are readily revealed by representation of molecular systems in an appropriate design space. Here, we first describe a generic approach to the construction of such a design space in which qualitatively distinct phenotypes can be identified and counted. Second, we show how the boundaries between these phenotypic regions provide a method of characterizing a system's tolerance to large changes in the values of its parameters. Third, we illustrate the approach for one of the most basic modules of biochemical networks and describe an associated design principle. Finally, we discuss the scaling of this approach to large systems.


Discriminability Tests for Visualization Effectiveness and Scalability.
The scalability of a particular visualization approach is limited by the ability for people to discern differences between plots made with different datasets. Ideally, when the data changes, the visualization changes in perceptible ways. This relation breaks down when there is a mismatch between the encoding and the character of the dataset being viewed. Unfortunately, visualizations are often designed and evaluated without fully exploring how they will respond to a wide variety of datasets. We explore the use of an image similarity measure, the Multi-Scale Structural Similarity Index (MS-SSIM), for testing the discriminability of a data visualization across a variety of datasets. MS-SSIM is able to capture the similarity of two visualizations across multiple scales, including low level granular changes and high level patterns. Significant data changes that are not captured by the MS-SSIM indicate visualizations of low discriminability and effectiveness. The measure's utility is demonstrated with two empirical studies. In the first, we compare human similarity judgments and MS-SSIM scores for a collection of scatterplots. In the second, we compute the discriminability values for a set of basic visualizations and compare them with empirical measurements of effectiveness. In both cases, the analyses show that the computational measure is able to approximate empirical results. Our approach can be used to rank competing encodings on their discriminability and to aid in selecting visualizations for a particular type of data distribution.


Long-range regulators of the lncRNA HOTAIR enhance its prognostic potential in breast cancer.
Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.


Felodipine in patients with chronic heart failure: discrepant haemodynamic and clinical effects.
Previous open studies have suggested that felodipine, a selective calcium antagonist and vasodilator, may be useful in the treatment of heart failure. A double blind placebo controlled crossover trial was therefore conducted to investigate the clinical and haemodynamic effects of felodipine in 15 patients with chronic ischaemic heart failure in New York Heart Association symptom class III. Felodipine significantly increased resting and exercise (25W bicycle ergometry) cardiac output without producing concomitant changes in resting or exercise heart rate or right and left ventricular filling pressures. Felodipine did not significantly improve symptom scores or exercise capacity in the group as a whole. It also resulted in significant fluid retention as shown by a rise in ankle circumference, body weight, and a fall in haematocrit. Further research is required to elucidate the mechanism that is responsible for the discrepancy between the haemodynamic and clinical effects of felodipine in patients with moderately severe heart failure.


Cytotoxicity and bioavailability studies on a decoction of Oldenlandia diffusa and its fractions separated by HPLC.
Oldenlandia diffusa is a traditional Chinese herbal remedy with known cytotoxic activity in vitro and in vivo. The aim of the study was to identify the most cytotoxic constituents of a water extract (a decoction is traditionally used as a treatment) by HPLC and activity-guided fractionation. The bioavailability of the decoction and certain fractions, and the mode of cell death induced by these mixtures, were also investigated. A decoction of O. diffusa was prepared and separated by HPLC into eleven fractions (F1-11) for testing on the growth of HL60 leukaemia cells; two of the most active fractions were also tested on Caco-2 colon cancer cells. Cell viability was measured by trypan blue exclusion, DNA content (Cyquant NF assay) and neutral red uptake. Evidence of apoptosis was gained from cells stained with the nuclear dye DAPI, and detection of cleaved poly (ADP-ribose) polymerase (PARP) by Western blot. Fraction 9 was found to be the most active fraction, and, along with the decoction, induced apoptosis. Cells stained with DAPI showed a decrease in cell size and nuclear fragmentation characteristic of apoptosis. Detection of cleaved PARP further confirmed induction of apoptosis by O. diffusa decoction and fraction 9. The bioavailability of O. diffusa was investigated by production of post-absorption samples using Caco-2 intestinal epithelial monolayers. Addition of post-absorption samples (taken from the basolateral side after 3h incubation with the decoction on the apical side) inhibited the growth of HL60 cells, and suggested a degree of bioavailability. The constituents in fraction 9 were further separated by HPLC and eight major compounds were identified by LC-MS: two of these were ursolic acid (UA) and its enantiomer oleanolic acid (OA). Concentrations of UA and OA in the decoction were then calculated by reference to the area of the peaks of UA and OA found in F9. The post-absorption sample of F9 contained six of the eight constituents in the original pre-absorption fraction 9. Taken together, the results suggest that certain constituents, possibly including ursolic/oleanolic acid, may be bioavailable and at sufficient concentration to induce apoptosis in cancer cells in vitro through a mechanism including the cleavage of PARP.


What do international pharmacoeconomic guidelines say about economic data transferability?
The objectives of this article were to assess the positions of the various national pharmacoeconomic guidelines on the transferability (or lack of transferability) of clinical and economic data and to review the methods suggested in the guidelines for addressing issues of transferability. A review of existing national pharmacoeconomic guidelines was conducted to assess recommendations on the transferability of clinical and economic data, whether there are important differences between countries, and whether common methodologies have been suggested to address key transferability issues. Pharmacoeconomic guidelines were initially identified through the ISPOR Web site. In addition, those national guidelines not included in the ISPOR Web site, but known to us, were also considered. Across 27 sets of guidelines, baseline risk and unit costs were uniformly considered to be of low transferability, while treatment effect was classified as highly transferable. Results were more variable for resource use and utilities, which were considered to have low transferability in 63% and 45% of cases, respectively. There were some differences between older and more recent guidelines in the treatment of transferability issues. A growing number of jurisdictions are using guidelines for the economic evaluation of pharmaceuticals. The recommendations in existing guidelines regarding the transferability of clinical and economic data are quite diverse. There is a case for standardization in dealing with transferability issues. One important step would be to update guidelines more frequently.


County-level pesticide use and risk of shortened gestation and preterm birth.
This study assesses the association between pesticide exposure in pregnancy, preterm birth (PTB) and shortened gestation. Pregnancy information was abstracted from the Centers for Disease Control (CDC) Non-Public Use Natality Datasets 1990-2005. Pesticide use in maternal county of residence was calculated using California Pesticide Use Reporting (PUR) data 1990-2005. Counties were ranked by pesticide use, and birth months were sorted by peak (May-June) or nonpeak (other months) pesticide use. Multivariate logistical regression models were used. Counties with higher pesticide use were associated with higher PTB (low 8.59 ± 0.11%, moderate 9.25 ± 0.07%, high 10.0 ± 0.06%, p's < 0.001) and shorter gestations (low 39.197 ± 0.014 weeks, moderate 39.126 ± 0.011 weeks, high 39.049 ± 0.011 weeks, p's < 0.001). Peak pesticide months were associated with higher PTB (10.01 ± 0.05% vs. 9.36 ± 0.05%, p < 0.001) and shorter gestations (39.069 ± 0.007 weeks vs. 39.122 ± 0.007 weeks, p < 0.001). The pesticide effect on shortened gestation and higher PTB was found in all racial groups. Pesticide use was highest for fungicides > insecticides > fumigants > herbicides > others. Each pesticide type was found to be associated with higher PTB and shorter gestation. PTB and shortened gestation were significantly associated with pesticide use in maternal county of residence regardless of race, gestation at birth, and in most risk categories.


Analysis of chronic obstructive pulmonary disease exacerbations with the dual bronchodilator QVA149 compared with glycopyrronium and tiotropium (SPARK): a randomised, double-blind, parallel-group study.
We evaluated the effect of dual, longacting inhaled bronchodilator treatment on exacerbations in patients with severe and very severe chronic obstructive pulmonary disease (COPD). In this parallel-group study, 2224 patients (aged ≥40 years, Global Initiative for Chronic Obstructive Lung Disease stages III-IV, and one or more moderate COPD exacerbation in the past year) were randomly assigned (1:1:1; via interactive voice response or web system; stratified for smoking status) to once-daily QVA149 (fixed-dose combination of indacaterol 110 μg and glycopyrronium 50 μg), glycopyrronium 50 μg, or tiotropium 18 μg for 64 weeks. Assignment to QVA149 and glycopyrronium was double-blind; tiotropium was open-label. Efficacy was assessed in all patients randomly assigned to treatment groups who received at least one dose of study drug; safety was assessed in all patients who received at least one dose whether or not they were assigned to a group. The primary objective was to show superiority of QVA149 versus glycopyrronium for rate of moderate to severe COPD exacerbations (defined by worsening symptoms and categorised by treatment requirements) during treatment. This completed trial is registered at ClinicalTrials.gov, NCT01120691. Between April 27, 2010, and July 11, 2012, 741 patients were randomly assigned to receive QVA149, 741 to receive glycopyrronium, and 742 to receive tiotropium (729, 739, and 737 patients, respectively, analysed for efficacy). QVA149 significantly reduced the rate of moderate to severe exacerbations versus glycopyrronium by 12% (annualised rate of exacerbations 0·84 [95% CI 0·75-0·94] vs 0·95 [0·85-1·06]; rate ratio 0·88, 95% CI 0·77-0·99, p=0·038). Adverse events (including exacerbations) were reported for 678 (93%) of 729 patients on QVA149, 694 (94%) of 740 on glycopyrronium, and 686 (93%) of 737 on tiotropium. Incidence of serious adverse events was similar between groups (167 [23%] patients on QVA149, 179 [24%] on glycopyrronium, and 165 [22%] on tiotropium); COPD worsening was the most frequent serious adverse event (107 [15%] patients on QVA149, 116 [16%] on glycopyrronium, 87 [12%] on tiotropium). The dual bronchodilator QVA149 was superior in preventing moderate to severe COPD exacerbations compared with the single longacting antimuscarinic bronchodilator glycopyrronium, with concomitant improvements in lung function and health status. These results indicate the potential of dual bronchodilation as a treatment option for patients with severe and very severe COPD. Novartis Pharma AG.


The Chlamydomonas cell wall: characterization of the wall framework.
The cell wall of the biflagellate alga Chlamydomonas reinhardtii is a multilayered, extracellular matrix composed of carbohydrates and 20-25 polypeptides. To learn more about the forces responsible for the integrity of this cellulose-deficient cell wall, we have begun studies to identify and characterize the framework of the wall and to determine the effects of the cell wall-degrading enzyme, lysin, on framework structure and protein composition. In these studies we used walls released into the medium by mating gametes. When isolated shed walls are degraded by exogenously added lysin, no changes are detected in the charge or molecular weight of the 20-25 wall proteins and glycoproteins when analyzed on one- and two-dimensional polyacrylamide gels, which suggests that degradation of these shed walls is due either to cleavage of peptide bonds very near the ends of polypeptides or that degradation occurs via a mechanism other than proteolysis. Incubation of walls with Sarkosyl-urea solutions removes most of the proteins and yields thin structures that appear to be the frameworks of the walls. Analysis by polyacrylamide gel electrophoresis shows that the frameworks are highly enriched in a polypeptide of Mr 100,000. Treatment of frameworks with lysin leads to their degradation, which indicates that this part of the wall is a substrate for the enzyme. Although lysin converts the Mr 100,000 polypeptide from an insoluble to a soluble form, there is no detectable change in Mr of the framework protein. Solubilization in the absence of lysin requires treatment with SDS and dithiothreitol at 100 degrees C. These results suggest that the Chlamydomonas cell wall is composed of two separate domains: one containing approximately 20 proteins held together by noncovalent interactions and a second domain, containing only a few proteins, which constitutes the framework of the wall. The result that shed walls can be solubilized by boiling in SDS-dithiothreitol indicates that disulfide linkages are critical for wall integrity. Using an alternative method for isolating walls from mechanically disrupted gametes, we have also shown that a wall-shaped portion of these unshed walls is insoluble under the same conditions in which shed walls are soluble. One interpretation of these results is that wall release during mating and the wall degradation that follows may involve distinct biochemical events.


Outside-host growth of pathogens attenuates epidemiological outbreaks.
Opportunist saprotrophic pathogens differ from obligatory pathogens due to their capability in host-independent growth in environmental reservoirs. Thus, the outside-host environment potentially influences host-pathogen dynamics. Despite the socio-economical importance of these pathogens, theory on their dynamics is practically missing. We analyzed a novel epidemiological model that couples outside-host density-dependent growth to host-pathogen dynamics. Parameterization was based on columnaris disease, a major hazard in fresh water fish farms caused by saprotrophic Flavobacterium columnare. Stability analysis and numerical simulations revealed that the outside-host growth maintains high proportion of infected individuals, and under some conditions can drive host extinct. The model can show stable or cyclic dynamics, and the outside-host growth regulates the frequency and intensity of outbreaks. This result emerges because the density-dependence stabilizes dynamics. Our analysis demonstrates that coupling of outside-host growth and traditional host-pathogen dynamics has profound influence on disease prevalence and dynamics. This also has implications on the control of these diseases.


The effect of high-dose steroid treatment used for the treatment of acute demyelinating diseases on endothelial and cardiac functions.
The cardiovascular effects of short-term high-dose steroid treatment (pulse steroid treatment) have not yet been clarified. We examined the short- and long-term effects of pulse steroid treatment in demyelinating diseases on endothelial and cardiac functions. In this prospective study, we included 35 patients (20 females and 15 males; mean age, 32.8±9.3 years) who were not treated with steroids and who were previously diagnosed with multiple sclerosis or neuromyelitis optica. Patients were evaluated before, 1 week after, and 3 months after the steroid treatment. Brachial artery flow-mediated relaxation and cardiac systolic/diastolic function were evaluated using echocardiography to assess physical examination results, carotid intima-media thickness, and endothelial function. There was no difference between biochemical values, systolic function, left ventricular dimensions, and carotid intima-media thicknesses in the three evaluation periods. There were significant increases in the body mass index, body weight, and systolic/diastolic blood pressure measurements at 1 week and 3 months after treatment (p<0.001). There was a significant decrease in brachial artery flow-mediated relaxation at 1 week and 3 months (1 versus 2, p=0.042; 1 versus 3, p=0.003). In Doppler measurements at 1 week and 3 months, there was an increase in mitral A velocity, IVRT, and EDT values and a decrease in the E/A ratio in line with diastolic dysfunction. Pulse steroid therapy used for demyelinating diseases deteriorated endothelial and left ventricular diastolic functions in the early and late periods. Future studies are needed to evaluate the development of cardiovascular mortality and morbidity in patients receiving this type of treatment.


Nutritional supplementation to reduce child aggression: a randomized, stratified, single-blind, factorial trial.
While some studies suggest that nutritional supplementation may reduce aggressive behavior in children, they have not examined whether its efficacy may be enhanced in conjunction with other treatment approaches. This study tests the hypothesis that a nutritional supplementation of omega-3, multivitamins, and minerals over 3 months, combined with cognitive behavior therapy, will reduce childhood aggression. In this randomized, single-blind, stratified, factorial trial, a high-risk community sample of 290 children aged 11-12 years were randomized into Nutrition only, cognitive behavioral therapy (CBT) only, Nutrition + CBT, and Control groups. The primary outcome measures of child- and parent-reported aggressive and antisocial behavior were collected at 0 months (baseline), 3 months (end of treatment), 6 months (3 months posttreatment), and 12 months (9 months posttreatment). The trial ('Healthy Brains & Behavior: Understanding and Treating Youth Aggression (HBB)' was registered at ClinicalTrials.gov at https://clinicaltrials.gov/ct2/show/NCT00842439 RESULTS: For child self-reports, children in the Nutrition only group showed reduced externalizing behavior compared to Controls at 3 months. At 6 months, the Nutrition + CBT group scored lower on externalizing behavior compared to both CBT only and Control groups. Findings were more in evidence for an Aggressive-Reactive form of antisocial behavior than for a Callous-Proactive form. Effect sizes were in the small-to-medium range (d = -.33 to -.37). Group differences were not sustained 9 months posttreatment, and no other effects were significant. Findings provide some limited support for the efficacy of omega-3, vitamin, and mineral supplementation in reducing aggressive behavior in children, and represent the first evaluation of nutritional supplements in conjunction with CBT.


Biomarker validation of dietary intervention in two multiethnic populations.
Intervention studies have been designed to change dietary and lifestyle factors associated with chronic diseases, but self-reported behavior change may incorporate intervention-related bias. This study examines plasma nutrient concentration and correlations with self-reports in the Healthy Directions intervention study. The Healthy Directions intervention studies were designed to increase multivitamin use, fruit and vegetable consumption, and physical activity in working-class, multiethnic populations. Participants in both studies completed interviewer-administered questionnaires that collected information on sociodemographic and health behavior characteristics. Postintervention blood samples were collected from 209 participants and pooled in pairs within study and within intervention group. We found significantly higher plasma concentrations of retinol (P = .01) and alpha-carotene (P = .03) in the intervention than in the usual care group. Self-reported multivitamin users had significantly higher concentrations of retinol (P < .001), beta-carotene (P = .02), and alpha-tocopherol (P < .001). Those who reported four or more fruit and vegetable servings per day had higher lutein and zeaxanthin (P = .05) and beta-cryptoxanthin (P = .05) concentrations than those consuming fewer. Plasma nutrient concentrations were associated with reported multivitamin use and fruit and vegetable intake, but the correlations were generally higher in the usual care group. We found significant postintervention differences in plasma carotenoid and tocopherol concentrations by treatment group, multivitamin use, and fruit and vegetable intake. However, because we only obtained postintervention blood samples, we were unable to assess preintervention-to-postintervention changes in plasma nutrients. Self-reported intakes were significantly correlated with plasma nutrient concentrations, but the strength of the correlations differed by group, suggesting some intervention-related bias in the questionnaire responses.


Predictive performance of comorbidity measures in administrative databases for diabetes cohorts.
The performance of comorbidity measures for predicting mortality in chronic disease populations and using ICD-9 diagnosis codes in administrative health data has been investigated in several studies, but less is known about predictive performance with ICD-10 data and for other health outcomes. This study investigated predictive performance of five comorbidity measures for population-based diabetes cohorts in administrative data. The objectives were to evaluate performance for: (a) disease-specific and general health outcomes, (b) data based on the ICD-9 and ICD-10 diagnoses, and (c) different age groups. Performance was investigated for heart attack, stroke, amputation, renal disease, hospitalization, and death in all-age and age-specific cohorts. Hospital records, physician billing claims, and prescription drug records from one Canadian province were used to identify diabetes cohorts and measure comorbidity. The data were analysed using multiple logistic regression models and summarized using measures of discrimination, accuracy, and fit. In Cohort 1 (n = 29,058), for which only ICD-9 diagnoses were recorded in administrative data, the Elixhauser index showed good or excellent prediction for amputation, renal disease, and death and performed better than the Charlson index. Number of diagnoses was a good predictor of hospitalization. Similar results were obtained for Cohort 2 (n = 41,925), in which both ICD-9 and ICD-10 diagnoses were recorded in administrative data, although predictive performance was sometimes higher. For age-specific models of mortality, the Elixhauser index resulted in the largest improvement in predictive performance in all but the youngest age group. Cohort age and the health outcome under investigation, but not the diagnosis coding system, may influence the predictive performance of comorbidity measure for studies about diabetes populations using administrative health data.


Multiple functions of Jab1 are required for early embryonic development and growth potential in mice.
Jab1 interacts with a variety of signaling molecules and regulates their stability in mammalian cells. As the fifth component of the COP9 signalosome (CSN) complex, Jab1 (CSN5) plays a central role in the deneddylation of the cullin subunit of the Skp1-Cullin-F box protein ubiquitin ligase complex. In addition, a CSN-independent function of Jab1 is suggested but is less well characterized. To elucidate the function of Jab1, we targeted the Jab1 locus by homologous recombination in mouse embryonic stem cells. Jab1-null embryos died soon after implantation. Jab1-/- embryonic cells, which lacked other CSN components, expressed higher levels of p27, p53, and cyclin E, resulting in impaired proliferation and accelerated apoptosis. Jab1 heterozygous mice were healthy and fertile but smaller than their wild-type littermates. Jab1+/- mouse embryonic fibroblast cells, in which the amount of Jab1-containing small subcomplex, but not that of CSN, was selectively reduced, proliferated poorly, showed an inefficient down-regulation of p27 during G1, and was delayed in the progression from G0 to S phase by 3 h compared with the wild-type cells. Most interestingly, in Jab1+/- mouse embryonic fibroblasts, the levels of cyclin E and deneddylated Cul1 were unchanged, and p53 was not induced. Thus, Jab1 controls cell cycle progression and cell survival by regulating multiple cell cycle signaling pathways.


Sequence and structure comparison suggest that methionine aminopeptidase, prolidase, aminopeptidase P, and creatinase share a common fold.
Amino acid sequence comparison suggests that the structure of Escherichia coli methionine aminopeptidase (EC 18.104.22.168) and the C-terminal domain of Pseudomonas putida creatinase (EC 126.96.36.199) are related. A detailed comparison of the three-dimensional folds of the two enzymes confirms this homology: with an approximately 260-residue chain segment, 218 C alpha atoms of the structures superimpose within 2.5 A; only 41 of these overlapping positions (i.e., 19%) feature identical amino acids in the two protein chains. Notwithstanding this striking correspondence in structure, methionine aminopeptidase binds and is stimulated by Co2+, while creatinase is not a metal-dependent enzyme. Searches of protein data banks using sequence and structure-based profiles reveal other enzymes, including aminopeptidase P (EC 18.104.22.168), prolidase (EC 18.104.22.168), and agropine synthase, that likely share the same "pita-bread" fold common to creatinase and methionine aminopeptidase.


High seroprevalence of Encephalitozoon cuniculi in pet rabbits in Japan.
Infection with Encephalitozoon cuniculi in rabbits frequently exists as a chronic, latent infection, and only a percentage of infected animals develop clinical disease. This study presents a seroepidemiological study of E. cunicucli infection in 337 pet rabbits collected from 20 prefectures in Japan in 2006 and 2007, using enzyme-linked immunosorbent assay (ELISA) capable of measuring IgG and IgM antibodies. These rabbits were divided into the following four groups: healthy and isolated rabbits (n=74, group I), healthy and companioned rabbits (n=121, group II), neurologically diseased rabbits (n=105, group III), and other diseased rabbits (n=37, group IV). Using ELISA for IgG antibodies, the highest detection rate, 81%, was seen in group III, the second highest, 75.2%, in group II, and the lowest, 29.7%, in group I, which was significantly different to the other groups except for group IV (43.2%). On the other hand, when ELISA was used for IgM antibody detection, 14-40% of rabbits in the four groups were also observed to have anti-E. cuniculi IgM. This study demonstrated high seroprevalence of E. cuniculi in not only neurologically diseased rabbits but also healthy and other diseased rabbits.


Regulation of protein kinase B and 4E-BP1 by oxidative stress in cardiac myocytes.
Stimulation of phosphatidylinositol 3'-kinase (PI3K) and protein kinase B (PKB) is implicated in the regulation of protein synthesis in various cells. One mechanism involves PI3K/PKB-dependent phosphorylation of 4E-BP1, which dissociates from eIF4E, allowing initiation of translation from the 7-methylGTP cap of mRNAs. We examined the effects of insulin and H(2)O(2) on this pathway in neonatal cardiac myocytes. Cardiac myocyte protein synthesis was increased by insulin, but was inhibited by H(2)O(2). PI3K inhibitors attenuated basal levels of protein synthesis and inhibited the insulin-induced increase in protein synthesis. Insulin or H(2)O(2) increased the phosphorylation (activation) of PKB through PI3K, but, whereas insulin induced a sustained response, the response to H(2)O(2) was transient. 4E-BP1 was phosphorylated in unstimulated cells, and 4E-BP1 phosphorylation was increased by insulin. H(2)O(2) stimulated dephosphorylation of 4E-BP1 by increasing protein phosphatase (PP1/PP2A) activity. This increased the association of 4E-BP1 with eIF4E, consistent with H(2)O(2) inhibition of protein synthesis. The effects of H(2)O(2) were sufficient to override the stimulation of protein synthesis and 4E-BP1 phosphorylation induced by insulin. These results indicate that PI3K and PKB are important regulators of protein synthesis in cardiac myocytes, but other factors, including phosphatase activity, modulate the overall response.


Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo.
Reprogramming somatic cells into an ESC-like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8–12 weeks. Differentiation was assessed by lineage-specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS-derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage-specific genes. Isolated implants of iPS cell-derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease.


Functional effects of spinocerebellar ataxia type 13 mutations are conserved in zebrafish Kv3.3 channels.
The zebrafish has been suggested as a model system for studying human diseases that affect nervous system function and motor output. However, few of the ion channels that control neuronal activity in zebrafish have been characterized. Here, we have identified zebrafish orthologs of voltage-dependent Kv3 (KCNC) K+ channels. Kv3 channels have specialized gating properties that facilitate high-frequency, repetitive firing in fast-spiking neurons. Mutations in human Kv3.3 cause spinocerebellar ataxia type 13 (SCA13), an autosomal dominant genetic disease that exists in distinct neurodevelopmental and neurodegenerative forms. To assess the potential usefulness of the zebrafish as a model system for SCA13, we have characterized the functional properties of zebrafish Kv3.3 channels with and without mutations analogous to those that cause SCA13. The zebrafish genome (release Zv8) contains six Kv3 family members including two Kv3.1 genes (kcnc1a and kcnc1b), one Kv3.2 gene (kcnc2), two Kv3.3 genes (kcnc3a and kcnc3b), and one Kv3.4 gene (kcnc4). Both Kv3.3 genes are expressed during early development. Zebrafish Kv3.3 channels exhibit strong functional and structural homology with mammalian Kv3.3 channels. Zebrafish Kv3.3 activates over a depolarized voltage range and deactivates rapidly. An amino-terminal extension mediates fast, N-type inactivation. The kcnc3a gene is alternatively spliced, generating variant carboxyl-terminal sequences. The R335H mutation in the S4 transmembrane segment, analogous to the SCA13 mutation R420H, eliminates functional expression. When co-expressed with wild type, R335H subunits suppress Kv3.3 activity by a dominant negative mechanism. The F363L mutation in the S5 transmembrane segment, analogous to the SCA13 mutation F448L, alters channel gating. F363L shifts the voltage range for activation in the hyperpolarized direction and dramatically slows deactivation. The functional properties of zebrafish Kv3.3 channels are consistent with a role in facilitating fast, repetitive firing of action potentials in neurons. The functional effects of SCA13 mutations are well conserved between human and zebrafish Kv3.3 channels. The high degree of homology between human and zebrafish Kv3.3 channels suggests that the zebrafish will be a useful model system for studying pathogenic mechanisms in SCA13.


Genetic variations associated with red hair color and fear of dental pain, anxiety regarding dental care and avoidance of dental care.
Red hair color is caused by variants of the melanocortin-1 receptor (MC1R) gene. People with naturally red hair are resistant to subcutaneous local anesthetics and, therefore, may experience increased anxiety regarding dental care. The authors tested the hypothesis that having natural red hair color, a MC1R gene variant or both could predict a patient's experiencing dental care-related anxiety and dental care avoidance. The authors enrolled 144 participants (67 natural red-haired and 77 dark-haired) aged 18 to 41 years in a cross-sectional observational study. Participants completed validated survey instruments designed to measure general and dental care-specific anxiety, fear of dental pain and previous dental care avoidance. The authors genotyped participants' blood samples to detect variants associated with natural red hair color. Eighty-five participants had MC1R gene variants (65 of the 67 red-haired participants and 20 of the 77 dark-haired participants) (P < .001). Participants with MC1R gene variants reported significantly more dental care-related anxiety and fear of dental pain than did participants with no MC1R gene variants. They were more than twice as likely to avoid dental care as were the participants with no MC1R gene variants, even after the authors controlled for general trait anxiety and sex. Dental care-related anxiety, fear of dental pain and avoidance of dental care may be influenced by genetic variations. Dentists should evaluate all patients, but especially those with naturally red hair, for dental care-related anxiety and use appropriate modalities to manage the patients' anxiety.


Imaging of osteoid osteoma with dynamic gadolinium-enhanced MR imaging.
To compare dynamic gadolinium-enhanced T1-weighted magnetic resonance (MR) imaging with nonenhanced T1-weighted and T2-weighted MR imaging and thin-section computed tomography (CT) for the demonstration of osteoid osteomas. The images of 11 patients with pathologically proven osteoid osteomas who underwent nonenhanced MR imaging, dynamic gadolinium-enhanced MR imaging, and CT were retrospectively reviewed. Images obtained with all three techniques were scored for conspicuity of the osteoid osteoma relative to the surrounding bone. Time-enhancement curves were generated from signal intensity measurements of these lesions and the adjacent bone marrow. The mean imaging scores of the four techniques were compared, and the statistical significance was calculated by using a linear model with terms for method and patient. Pairwise comparisons were made by using the Tukey-Kramer adjustment for multiple comparisons. Compared with CT, dynamic gadolinium-enhanced MR imaging demonstrated the osteoid osteoma equally well in eight of 11 patients and with better conspicuity in three of 11 patients, although this difference was not statistically significant (P =.69). The dynamic gadolinium-enhanced MR images demonstrated the osteoid osteomas significantly better than the nonenhanced T1-weighted (P <.001) and T2-weighted (P <.001) MR images. On the dynamic gadolinium-enhanced MR images, nine (82%) of 11 patients had peak enhancement of the osteoid osteoma in the arterial phase with early partial washout, compared with slower, progressive enhancement of the adjacent marrow. This resulted in greatest lesion to marrow contrast material enhancement in the arterial phase. One osteoid osteoma had peak enhancement in the venous phase, and one showed progressive enhancement through all phases to 150 seconds. Osteoid osteomas can be imaged with greater conspicuity by using dynamic gadolinium-enhanced instead of nonenhanced MR imaging and with conspicuity equal to or better than that obtained with thin-section CT.


Gilbert's syndrome and the risk of death: a population-based cohort study.
Gilbert's syndrome is a common familial hyperbilirubinemia that may reduce the risk of various age-related diseases because of the antioxidant properties of bilirubin. We conducted a large cohort study using The Health Improvement Network primary care database and compared all-cause mortality rates in those with and without Gilbert's syndrome. Mortality rates in patients with a diagnosis of Gilbert's syndrome and raised bilirubin level (n = 4266) were compared with those of patients with similar characteristics but with normal bilirubin levels (n = 21 968). Multivariate Poisson regression was also used to estimate adjusted mortality rate ratios. During the 350 000 PYs of follow up across the Gilbert's and comparison cohorts, there were 1174 deaths. Mortality rates were 24/10 000 PYs in the Gilbert's cohort versus 50/10 000 PYs in the comparison cohort. Mortality rates were around half in patients with Gilbert's syndrome after accounting for sociodemographics and general health indicators (adjusted mortality rate ratio: 0.5 [95% confidence interval; 0.4-0.7; P < 0.001]). Mortality rates observed for people with Gilbert's syndrome in the general population are almost half those of people without evidence of Gilbert's syndrome.


Perioperative beta-blockers for preventing surgery-related mortality and morbidity.
Randomized controlled trials have yielded conflicting results regarding the ability of beta-blockers to influence perioperative cardiovascular morbidity and mortality. Thus routine prescription of these drugs in unselected patients remains a controversial issue. The objective of this review was to systematically analyse the effects of perioperatively administered beta-blockers for prevention of surgery-related mortality and morbidity in patients undergoing any type of surgery while under general anaesthesia. We identified trials by searching the following databases from the date of their inception until June 2013: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials (CENTRAL), Biosis Previews, CAB Abstracts, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Derwent Drug File, Science Citation Index Expanded, Life Sciences Collection, Global Health and PASCAL. In addition, we searched online resources to identify grey literature. We included randomized controlled trials if participants were randomly assigned to a beta-blocker group or a control group (standard care or placebo). Surgery (any type) had to be performed with all or at least a significant proportion of participants under general anaesthesia. Two review authors independently extracted data from all studies. In cases of disagreement, we reassessed the respective studies to reach consensus. We computed summary estimates in the absence of significant clinical heterogeneity. Risk ratios (RRs) were used for dichotomous outcomes, and mean differences (MDs) were used for continuous outcomes. We performed subgroup analyses for various potential effect modifiers. We included 89 randomized controlled trials with 19,211 participants. Six studies (7%) met the highest methodological quality criteria (studies with overall low risk of bias: adequate sequence generation, adequate allocation concealment, double/triple-blinded design with a placebo group, intention-to-treat analysis), whereas in the remaining trials, some form of bias was present or could not be definitively excluded (studies with overall unclear or high risk of bias). Outcomes were evaluated separately for cardiac and non-cardiac surgery. CARDIAC SURGERY (53 trials)We found no clear evidence of an effect of beta-blockers on the following outcomes.• All-cause mortality: RR 0.73, 95% CI 0.35 to 1.52, 3783 participants, moderate quality of evidence.• Acute myocardial infarction (AMI): RR 1.04, 95% CI 0.71 to 1.51, 3553 participants, moderate quality of evidence.• Myocardial ischaemia: RR 0.51, 95% CI 0.25 to 1.05, 166 participants, low quality of evidence.• Cerebrovascular events: RR 1.52, 95% CI 0.58 to 4.02, 1400 participants, low quality of evidence.• Hypotension: RR 1.54, 95% CI 0.67 to 3.51, 558 participants, low quality of evidence.• Bradycardia: RR 1.61, 95% CI 0.97 to 2.66, 660 participants, low quality of evidence.• Congestive heart failure: RR 0.22, 95% CI 0.04 to 1.34, 311 participants, low quality of evidence.Beta-blockers significantly reduced the occurrence of the following endpoints.• Ventricular arrhythmias: RR 0.37, 95% CI 0.24 to 0.58, number needed to treat for an additional beneficial outcome (NNTB) 29, 2292 participants, moderate quality of evidence.• Supraventricular arrhythmias: RR 0.44, 95% CI 0.36 to 0.53, NNTB six, 6420 participants, high quality of evidence.• On average, beta-blockers reduced length of hospital stay by 0.54 days (95% CI -0.90 to -0.19, 2450 participants, low quality of evidence). NON-CARDIAC SURGERY (36 trials)We found a potential increase in the occurrence of the following outcomes with the use of beta-blockers.• All-cause mortality: RR 1.24, 95% CI 0.99 to 1.54, 11,463 participants, low quality of evidence.Whereas no clear evidence of an effect was noted when all studies were analysed, restricting the meta-analysis to low risk of bias studies revealed a significant increase in all-cause mortality with the use of beta-blockers: RR 1.27, 95% CI 1.01 to 1.59, number needed to treat for an additional harmful outcome (NNTH) 189, 10,845 participants.• Cerebrovascular events: RR 1.59, 95% CI 0.93 to 2.71, 9150 participants, low quality of evidence.Whereas no clear evidence of an effect was found when all studies were analysed, restricting the meta-analysis to low risk of bias studies revealed a significant increase in cerebrovascular events with the use of beta-blockers: RR 2.09, 95% CI 1.14 to 3.82, NNTH 255, 8648 participants.Beta-blockers significantly reduced the occurrence of the following endpoints.• AMI: RR 0.73, 95% CI 0.61 to 0.87, NNTB 72, 10,958 participants, high quality of evidence.• Myocardial ischaemia: RR 0.43, 95% CI 0.27 to 0.70, NNTB seven, 1028 participants, moderate quality of evidence.• Supraventricular arrhythmias: RR 0.72, 95% CI 0.56 to 0.92, NNTB 111, 8794 participants, high quality of evidence.Beta-blockers significantly increased the occurrence of the following adverse events.• Hypotension: RR 1.50, 95% CI 1.38 to 1.64, NNTH 15, 10,947 participants, high quality of evidence.• Bradycardia: RR 2.24, 95% CI 1.49 to 3.35, NNTH 18, 11,083 participants, moderate quality of evidence.We found no clear evidence of an effect of beta-blockers on the following outcomes.• Ventricular arrhythmias: RR 0.64, 95% CI 0.30 to 1.33, 526 participants, moderate quality of evidence.• Congestive heart failure: RR 1.17, 95% CI 0.93 to 1.47, 9223 participants, moderate quality of evidence.• Length of hospital stay: mean difference -0.27 days, 95% CI -1.29 to 0.75, 601 participants, low quality of evidence. According to our findings, perioperative application of beta-blockers still plays a pivotal role in cardiac surgery , as they can substantially reduce the high burden of supraventricular and ventricular arrhythmias in the aftermath of surgery. Their influence on mortality, AMI, stroke, congestive heart failure, hypotension and bradycardia in this setting remains unclear.In non-cardiac surgery, evidence from low risk of bias trials shows an increase in all-cause mortality and stroke with the use of beta-blockers. As the quality of evidence is still low to moderate, more evidence is needed before a definitive conclusion can be drawn. The substantial reduction in supraventricular arrhythmias and AMI in this setting seems to be offset by the potential increase in mortality and stroke.


Hyperlipidemia during gestational diabetes and its relation with maternal and offspring complications.
lipid profile suffers adaptive changes during pregnancy due to estrogen stimulation and insulin resistance. Several relations have been suggested between maternal lipid profile, glucose tolerance, endothelial cell dysfunction and long-term cardiovascular risk; the effects of maternal lipid profile metabolism in fetal growth are also inconclusive. Since a regular evaluation and follow-up of lipid profile during pregnancy has not been established yet, we aimed to evaluate the incidence of dyslipidemia in patients with gestational diabetes (GDM) and analyze some putative relations with pregnancy, offspring complications and maternal metabolic syndrome parameters determined three and twelve months after delivery. two hundred and fifty patients with GDM were included. Full medical history, offspring characteristics, lipid profile and maternal variables of metabolic syndrome were evaluated during pregnancy and three- and twelve-months after delivery. The incidence of dyslipidemia during pregnancy was determined using two different classifications. lower plasma HDL and hypertriglyceridemia were the most current disorders; prematurity or birth weight were not correlated with dyslipidemia. During pregnancy, the lipid-related parameter that better predicted the risk of offspring macrosomia was triglycerides (TG). High TG three months after delivery were correlated to macrosomia and metabolic syndrome variables before and after pregnancy (three and twelve months). TG during pregnancy is the parameter that best predicts the risk of macrosomia and is related to increased metabolic risk after delivery. The evaluation of lipid profile and other metabolic variables during pregnancy and after delivery is required to early diagnose cardiovascular risk factors, especially in high risk population.


Relationship of CYP2D6, CYP3A, POR, and ABCB1 genotypes with galantamine plasma concentrations.
The frequently prescribed antidementia drug galantamine is extensively metabolized by the enzymes cytochrome P450 (CYP) 2D6 and CYP3A and is a substrate of the P-glycoprotein. We aimed to study the relationship between genetic variants influencing the activity of these enzymes and transporters with galantamine steady state plasma concentrations. In this naturalistic cross-sectional study, 27 older patients treated with galantamine were included. The patients were genotyped for common polymorphisms in CYP2D6, CYP3A4/5, POR, and ABCB1, and galantamine steady state plasma concentrations were determined. The CYP2D6 genotype seemed to be an important determinant of galantamine pharmacokinetics, with CYP2D6 poor metabolizers presenting 45% and 61% higher dose-adjusted galantamine plasma concentrations than heterozygous and homozygous CYP2D6 extensive metabolizers (median 2.9 versus 2.0 ng/mL · mg, P = 0.025, and 1.8 ng/mL · mg, P = 0.004), respectively. The CYP2D6 genotype significantly influenced galantamine plasma concentrations. The influence of CYP2D6 polymorphisms on the treatment efficacy and tolerability should be further investigated.


The diagnostic accuracy and utility of a B-type natriuretic peptide test in a community population of patients with suspected heart failure.
National guidelines suggest the use of natriuretic peptides in suspected heart failure but there have been no studies comparing assays in primary care. To test and compare the diagnostic accuracy and utility of B-type natriuretic peptide (BNP) and N-terminal B-type natriuretic peptide (NT proBNP) in diagnosing heart failure due to left ventricular systolic dysfunction in patients with suspected heart failure referred by GPs to one-stop diagnostic clinics. Community cohort, prospective, diagnostic accuracy study. One-stop diagnostic clinics in Darlington Memorial and Bishop Auckland General Hospitals and general practices in South Durham. Two hundred and ninety-seven consecutive patients with symptoms and signs suggestive of heart failure referred from general practice. The study measured sensitivity, specificity, positive and negative predictive values (PPV, NPV), and area under receiver operating characteristic curve for BNP (near patient assay) and NT proBNP (laboratory assay) in diagnosis of heart failure due to left ventricular systolic dysfunction. The NPV of both assays was determined as a potential method of reducing the number of referrals for echocardiography. One hundred and fourteen of the 297 patients had left ventricular systolic dysfunction (38%). At the manufacturer's recommended cut-off of 100 pg/ml BNP gave a NPV of 82%. BNP performed better at a cut-off of 40 pg/ml with a NPV of 88%. At a cut-off of 150 pg/ml, NT proBNP gave a NPV of 92%. Using cut-offs of 40 pg/ml and 150 pg/ml for BNP and NT pro-BNP, respectively, could have prevented 24% and 25% of referrals to the clinic, respectively. In this setting, NT pro-BNP performed marginally better than BNP, and would be easier to use practically in primary care. A satisfactory cut-off has been identified, which needs validating in general practice. NT pro-BNP could be used to select referrals to a heart failure clinic or for echocardiography. This process needs testing in real-life general practice.


The next generation of site-based long-term ecological monitoring: Linking essential biodiversity variables and ecosystem integrity.
Global change effects on biodiversity and human wellbeing call for improved long-term environmental data as a basis for science, policy and decision making, including increased interoperability, multifunctionality, and harmonization. Based on the example of two global initiatives, the International Long-Term Ecological Research (ILTER) network and the Group on Earth Observations Biodiversity Observation Network (GEO BON), we propose merging the frameworks behind these initiatives, namely ecosystem integrity and essential biodiversity variables, to serve as an improved guideline for future site-based long-term research and monitoring in terrestrial, freshwater and coastal ecosystems. We derive a list of specific recommendations of what and how to measure at a monitoring site and call for an integration of sites into co-located site networks across individual monitoring initiatives, and centered on ecosystems. This facilitates the generation of linked comprehensive ecosystem monitoring data, supports synergies in the use of costly infrastructures, fosters cross-initiative research and provides a template for collaboration beyond the ILTER and GEO BON communities.


The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata.
Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.


Familial episodic limb pain in kindreds with novel Nav1.9 mutations.
We previously performed genetic analysis in six unrelated families with infantile limb pain episodes, characterized by cold-induced deterioration and mitigation in adolescence, and reported two new mutations p.R222H/S in SCN11A responsible for these episodes. As no term described this syndrome (familial episodic pain: FEP) in Japanese, we named it as"". In the current study, we recruited an additional 42 new unrelated Japanese FEP families, between March 2016 and March 2018, and identified a total of 11 mutations in SCN11A: p.R222H in seven families, and p.R225C, p.F814C, p.F1146S, or p.V1184A, in independent families. A founder mutation, SCN11A p.R222H was confirmed to be frequently observed in patients with FEP in the Tohoku region of Japan. We also identified two novel missense variants of SCN11A, p.F814C and p.F1146S. To evaluate the effects of these latter two mutations, we generated knock-in mouse models harboring p.F802C (F802C) and p.F1125S (F1125S), orthologues of the human p.F814C and p.F1146S, respectively. We then performed electrophysiological investigations using dorsal root ganglion neurons dissected from the 6-8 week-old mice. Dissected neurons of F802C and F1125S mice showed increased resting membrane potentials and firing frequency of the action potentials (APs) by high input-current stimulus compared with WT mice. Furthermore, the firing probability of evoked APs increased in low stimulus input in F1125S mice, whereas several AP parameters and current threshold did not differ significantly between either of the mutations and WT mice. These results suggest a higher level of excitability in the F802C or F1125S mice than in WT, and indicate that these novel mutations are gain of function mutations. It can be expected that a considerable number of potential patients with FEP may be the result of gain of function SCN11A mutations.


Measurement of PMD tolerance in 40-Gb/s polarization-multiplexed RZ-DQPSK.
We experimentally investigate the first-order polarization-mode dispersion (PMD) tolerance of two polarization-multiplexed (POLMUX) RZ-DQPSK signals at overall 40 Gb/s. The polarization demultiplexing is enabled by an automatic endless polarization stabilizer. Time-interleaving the two orthogonally polarized RZ-DQPSK signals minimizes the crosstalk due to the non-ideal polarization stabilization, while it represents the worstcase for the PMD-induced crosstalk. Bit-error rate measurements are performed both in back-to-back and after 25-km standard single-mode fiber. The PMD tolerance is evaluated as a function of the instantaneous differential group delay, introduced by a first-order PMD emulator. 40-Gb/s POLMUX RZ-DQPSK is more sensitive to PMD than single-polarization 20-Gb/s DQPSK, while it is more PMD-tolerant than 40-Gb/s NRZ-OOK. Besides, its chromatic dispersion robustness is similar to the single-polarization 20-Gb/s DQPSK. The combination of POLMUX and DQPSK is therefore very promising in view of transmission systems at high bit-rate.


Impacts on terrestrial biodiversity of moving from a 2°C to a 1.5°C target.
We applied a recently developed tool to examine the reduction in climate risk to biodiversity in moving from a 2°C to a 1.5°C target. We then reviewed the recent literature examining the impact of (a) land-based mitigation options and (b) land-based greenhouse gas removal options on biodiversity. We show that holding warming to 1.5°C versus 2°C can significantly reduce the number of species facing a potential loss of 50% of their climatic range. Further, there would be an increase of 5.5-14% of the globe that could potentially act as climatic refugia for plants and animals, an area equivalent to the current global protected area network. Efforts to meet the 1.5°C target through mitigation could largely be consistent with biodiversity protection/enhancement. For impacts of land-based greenhouse gas removal technologies on biodiversity, some (e.g. soil carbon sequestration) could be neutral or positive, others (e.g. bioenergy with carbon capture and storage) are likely to lead to conflicts, while still others (e.g. afforestation/reforestation) are context-specific, when applied at scales necessary for meaningful greenhouse gas removal. Additional effort to meet the 1.5°C target presents some risks, particularly if inappropriately managed, but it also presents opportunities.This article is part of the theme issue 'The Paris Agreement: understanding the physical and social challenges for a warming world of 1.5°C above pre-industrial levels'.


Organochlorines and risk of non-Hodgkin lymphoma.
Organochlorine chemicals and polychlorinated biphenyls (PCBs) have been suspected as possible risk factors for non-Hodgkin lymphoma (NHL). We investigated PCBs and organochlorine pesticides and risk of NHL in a population-based case-control study in British Columbia, Canada. Congeners of PCBs (including dioxinlike congeners) and pesticides or pesticide metabolites were measured in plasma of 422 pretreatment cases and 460 control subjects. This is so far the largest study to examine organochlorines in plasma to date. Several dioxin-like PCB congeners were associated with increased risk of NHL, including dioxin-like PCB nos. 118 and 156 with odds ratios (OR) for the highest versus lowest quartile between 1.6 and 1.8. Several non-dioxin-like congeners also showed significant associations. The PCB congener with the strongest association was no. 180 with an OR for the highest versus the lowest quartile of 1.83 (95% confidence interval = 1.18-2.84). Six pesticide analytes also showed a significant association with NHL; beta-hexachlorocyclohexane, p,p'-DDE, hexachlorobenzene, mirex, oxychlordane and trans-nonachlor. The strongest association was found for oxychlordane, a metabolite of the pesticide chlordane (highest vs. lowest quartile OR = 2.68, 95% confidence interval = 1.69-4.24). Our results provide further evidence that organochlorines contribute to NHL risk.


Complete genome sequence and transcriptomic analysis of a novel marine strain Bacillus weihaiensis reveals the mechanism of brown algae degradation.
A novel marine strain representing efficient degradation ability toward brown algae was isolated, identified, and assigned to Bacillus weihaiensis Alg07. The alga-associated marine bacteria promote the nutrient cycle and perform important functions in the marine ecosystem. The de novo sequencing of the B. weihaiensis Alg07 genome was carried out. Results of gene annotation and carbohydrate-active enzyme analysis showed that the strain harbored enzymes that can completely degrade alginate and laminarin, which are the specific polysaccharides of brown algae. We also found genes for the utilization of mannitol, the major storage monosaccharide in the cell of brown algae. To understand the process of brown algae decomposition by B. weihaiensis Alg07, RNA-seq transcriptome analysis and qRT-PCR were performed. The genes involved in alginate metabolism were all up-regulated in the initial stage of kelp degradation, suggesting that the strain Alg07 first degrades alginate to destruct the cell wall so that the laminarin and mannitol are released and subsequently decomposed. The key genes involved in alginate and laminarin degradation were expressed in Escherichia coli and characterized. Overall, the model of brown algae degradation by the marine strain Alg07 was established, and novel alginate lyases and laminarinase were discovered.


Nyctiphanes couchii as intermediate host for Rhadinorhynchus sp. (Acanthocephala, Echinorhynchidae) from NW Iberian Peninsula waters.
In the mesozooplanktonic community of the coastal upwelling system of the Ría de Vigo (NW Spain), the euphausiid Nyctiphanes couchii has been identified for the first time in temperate waters of the NE Atlantic as the intermediate host for cystacanths of Rhadinorhynchus sp. Parasites were identified using morphological characters described in 20 cystacanths. The hooks of the proboscis were arranged in 14 rows of 26 hooks each, while the hooks of the basal circle were only slightly erected and were longer than remaining spines. A maximum-likelihood estimation (ML) tree inferred from the 18S rRNA data set of Palaeacantocephala revealed that our specimens belong to a highly supported clade with Rhadinorhynchus sp., Pararhadinorhynchus sp. and Transvena annulospinosa. Nonetheless, our morphological and phylogenetic analyses suggested that the status of Rhadinorhynchus pristis should be re-examined. The prevalences of parasites were 0.0019% and 0.0001% for frontal and coastal summer communities, and 0.0068% and 0.0008% for coastal and oceanic autumn communities, respectively. The presence of these cystacanths in different mesozooplankton communities throughout the study suggests that the recruitment of parasites may be affected by the oceanography.


Superresolution imaging via ptychography.
Coherent diffractive imaging of objects is made considerably more practicable by using ptychography, where a set of diffraction patterns replaces a single measurement and introduces a high degree of redundancy into the recorded data. Here we demonstrate that this redundancy allows diffraction patterns to be extrapolated beyond the aperture of the recording device, leading to superresolved images, improving the limit on the finest feature separation by more than a factor of 3.


Characteristics of alanine: glyoxylate aminotransferase from Saccharomyces cerevisiae, a regulatory enzyme in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates.
Alanine: glyoxylate aminotransferase (EC 18.104.22.168), which is involved in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates in Saccharomyces cerevisiae, was highly purified and characterized. The enzyme had Mr about 80 000, with two identical subunits. It was highly specific for L-alanine and glyoxylate and contained pyridoxal 5'-phosphate as cofactor. The apparent Km values were 2.1 mM and 0.7 mM for L-alanine and glyoxylate respectively. The activity was low (10 nmol/min per mg of protein) with glucose as sole carbon source, but was remarkably high with ethanol or acetate as carbon source (930 and 430 nmol/min per mg respectively). The transamination of glyoxylate is mainly catalysed by this enzyme in ethanol-grown cells. When glucose-grown cells were incubated in medium containing ethanol as sole carbon source, the activity markedly increased, and the increase was completely blocked by cycloheximide, suggesting that the enzyme is synthesized de novo during the incubation period. Similarity in the amino acid composition was observed, but immunological cross-reactivity was not observed among alanine: glyoxylate aminotransferases from yeast and vertebrate liver.


Randomized Trial of Nocturnal Oxygen in Chronic Obstructive Pulmonary Disease.
Long-term oxygen therapy improves survival in patients with chronic obstructive pulmonary disease (COPD) and chronic severe daytime hypoxemia. However, the efficacy of oxygen therapy for the management of isolated nocturnal hypoxemia is uncertain. We designed this double-blind, placebo-controlled, randomized trial to determine, in patients with COPD who have nocturnal arterial oxygen desaturation without qualifying for long-term oxygen therapy, whether nocturnal oxygen provided for a period of 3 to 4 years would decrease mortality or the worsening of disease such that patients meet current specifications for long-term oxygen therapy. Patients with an oxygen saturation of less than 90% for at least 30% of the recording time on nocturnal oximetry were assigned, in a 1:1 ratio, to receive either nocturnal oxygen or ambient air from a sham concentrator (placebo). The primary outcome was a composite of death from any cause or a requirement for long-term oxygen therapy as defined by the Nocturnal Oxygen Therapy Trial (NOTT) criteria in the intention-to-treat population. Recruitment was stopped prematurely because of recruitment and retention difficulties after 243 patients, of a projected 600, had undergone randomization at 28 centers. At 3 years of follow-up, 39.0% of the patients assigned to nocturnal oxygen (48 of 123) and 42.0% of those assigned to placebo (50 of 119) met the NOTT-defined criteria for long-term oxygen therapy or had died (difference, -3.0 percentage points; 95% confidence interval, -15.1 to 9.1). Our underpowered trial provides no indication that nocturnal oxygen has a positive or negative effect on survival or progression to long-term oxygen therapy in patients with COPD. (Funded by the Canadian Institutes of Health Research; INOX ClinicalTrials.gov number, NCT01044628.).


Hyperplasia of lymphatic vessels in VEGF-C transgenic mice.
No growth factors specific for the lymphatic vascular system have yet been described. Vascular endothelial growth factor (VEGF) regulates vascular permeability and angiogenesis, but does not promote lymphangiogenesis. Overexpression of VEGF-C, a ligand of the VEGF receptors VEGFR-3 and VEGFR-2, in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement. Thus, VEGF-C induces selective hyperplasia of the lymphatic vasculature, which is involved in the draining of interstitial fluid and in immune function, inflammation, and tumor metastasis. VEGF-C may play a role in disorders involving the lymphatic system and may be of potential use in therapeutic lymphangiogenesis.


Interaction between ingested nutrients and gut endocrine cells in patients with irritable bowel syndrome (review).
Several endocrine cell abnormalities have been reported in different segments of the gastrointestinal tract of patients with irritable bowel syndrome (IBS). These cells have specialized microvilli that project into the lumen; they function as sensors for the gut contents and respond to luminal stimuli (mostly ingested nutrients) by releasing hormones into the lamina propria, where they exert their effects via a paracrine/endocrine mode of action. Certain food items trigger the symptoms experienced by IBS patients, including those rich in fermentable oligo-, di- and monosaccharides, and polyols (FODMAPs). In this review, we present the argument that the effects of both FODMAPs and the proportional intake of proteins, fats and carbohydrates on IBS symptoms may be caused by an interaction with the gut endocrine cells. Since the gut hormones control and regulate gastrointestinal motility and sensation, this interaction may be responsible for abnormal gastrointestinal motility and the visceral hypersensitivity observed in these patients. There is no consistent evidence that IBS patients suffer from food allergy. The role of gluten intolerance in the development of IBS symptoms in these patients remains a matter of controversy. Individual guidance on food management, which includes restrictions in the intake of FODMAP-rich foods and testing diets with different proportions of proteins, fats and carbohydrates has been found to reduce the symptoms, improve the quality of life, and make the habitual diet of IBS patients more healthy.


Effects of synthetic para-nonylphenol isomers administered chronically throughout pregnancy and lactation on reproductive system of mouse pups.
The aim of this study was to analyze the effects of synthetic para-nonylphenol isomers administered chronically throughout pregnancy and lactation on the reproductive system of mouse pups. The two synthetic isomers used in this study showed higher (3E22NP) or lower (44NP) estrogen receptor (ER) binding activity on in vitro yeast assay than a commercial NP (NPmix). Female mice were implanted with a tube filled with one of three NPs and estradiol-17 β (E2) before mating. The tube was kept in the mice throughout pregnancy and lactation period, until their pups had weaned at 28 days of age (PND 28). The data indicated that in males, NPs decreased body weight in some dose groups on PND28 and increased weights of testes and epididymides. However, the rate of seminiferous tubules having elongate spermatids (steps 10-16) decreased significantly in NPs and E2 treated groups, except for the 44NP groups. In female mice, NPs and E2 increased weights of ovaries, uterus and vagina in the pups in some dose groups on PND 28. However, there were no differences in the day of vaginal opening and numbers of corpus lutea. The present study demonstrates that the effects of these two isomers of NP on the pup reproduction at PND 28 are not quite different, despite them showing different levels of ER binding activity using in vitro yeast assay.


Monocytic parameters in patients with dysthymia versus major depression.
The studies on monocytic function during depression are controversial. A better knowledge of affective disorders may improve the differential diagnosis of depression subtypes. Our goals are to examine if there are differences in monocytic function in patients with major depression and dysthymia. Twenty-two depressed patients (12 dysthymia and 10 major depression) and 15 healthy controls participated in the study. We analyzed monocyte count, monocyte subsets (CD14+, CD16+, and HLA class-II+), respiratory burst activity, phagocytic index and the interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNFalpha) production. Depressed patients showed elevated IL-1beta (P<0.05) and IL-6 (P<0.01), elevated monocytic respiratory burst activity (P<0.01); and reduced surface molecule expression HLA class-II and phagocytosis (P<0.01) compared with controls. We found no differences in any monocytic parameters between dysthymia and major depression. The small sample size and the short wash-out reduce the reliability of the results. Major depression and dysthymia show similar signs of both monocytic activation and suppression. These alterations may be due to the depressive syndrome and not to the characteristics of depression subtypes studied.


Coding and decoding libraries of sequence-defined functional copolymers synthesized via photoligation.
Designing artificial macromolecules with absolute sequence order represents a considerable challenge. Here we report an advanced light-induced avenue to monodisperse sequence-defined functional linear macromolecules up to decamers via a unique photochemical approach. The versatility of the synthetic strategy-combining sequential and modular concepts-enables the synthesis of perfect macromolecules varying in chemical constitution and topology. Specific functions are placed at arbitrary positions along the chain via the successive addition of monomer units and blocks, leading to a library of functional homopolymers, alternating copolymers and block copolymers. The in-depth characterization of each sequence-defined chain confirms the precision nature of the macromolecules. Decoding of the functional information contained in the molecular structure is achieved via tandem mass spectrometry without recourse to their synthetic history, showing that the sequence information can be read. We submit that the presented photochemical strategy is a viable and advanced concept for coding individual monomer units along a macromolecular chain.


Antiatherogenic components of olive oil.
Olive oil is the principal source of fat in the Mediterranean diet, which has been associated with a lower incidence of coronary heart disease and certain cancers. Olive oil is characterized by a high proportion of monounsaturated oleic acid, but the main peculiarity of extra-virgin oil is the presence of remarkable quantities of phenolic compounds, notably hydroxytyrosol and oleuropein, that provide high stability and strong taste. Recently, several studies have demonstrated that olive oil phenolics are powerful antioxidants, both in vitro and in vivo, and exert additional potent biologic activities that could partially account for the observed cardioprotective effects of the Mediterranean diet.


The SESA network links duplication of the yeast centrosome with the protein translation machinery.
The yeast spindle pole body (SPB), the functional equivalent of mammalian centrosome, duplicates in G1/S phase of the cell cycle and then becomes inserted into the nuclear envelope. Here we describe a link between SPB duplication and targeted translation control. When insertion of the newly formed SPB into the nuclear envelope fails, the SESA network comprising the GYF domain protein Smy2, the translation inhibitor Eap1, the mRNA-binding protein Scp160 and the Asc1 protein, specifically inhibits initiation of translation of POM34 mRNA that encodes an integral membrane protein of the nuclear pore complex, while having no impact on other mRNAs. In response to SESA, POM34 mRNA accumulates in the cytoplasm and is not targeted to the ER for cotranslational translocation of the protein. Reduced level of Pom34 is sufficient to restore viability of mutants with defects in SPB duplication. We suggest that the SESA network provides a mechanism by which cells can regulate the translation of specific mRNAs. This regulation is used to coordinate competing events in the nuclear envelope.


The past, present and future of childhood malaria mortality in Africa.
During the past few years, there has been a historic series of declarations of renewed commitment to malaria control in Africa. Whether the burden of malaria is increasing in Africa is a moot point. This article attempts to re-construct the evidence for the trends in childhood mortality as a result of Plasmodium falciparum infection over the last century in Africa.


Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms.
SIRT3 is identified as the major mitochondrial deacetylase. Two distinct isoforms of the murine SIRT3 have been identified with the short isoform having no recognizable mitochondrial localization sequence (MLS) and the long isoform having a putative MLS. A recent study questions the mitochondrial deacetylase activity of this short isoform. In contrast, the long isoform has been shown to be predominantly mitochondrial with robust deacetylase activity. In this study, we investigate whether the amino-terminus of the long SIRT3 isoform is a legitimate MLS and evaluate in-situ mitochondrial deacetylase activity of both isoforms. We confirm the presence of long and short isoforms in murine liver and kidney. The long isoform is generated via intra-exon splicing creating a frame-shift to expose a novel upstream translation start site. Mitochondrial localization is significantly more robust following transfection of the long compared with the short isoform. Insertion of this alternatively spliced novel 5' sequence upstream of a GFP-reporter plasmid shows greater than 80% enrichment in mitochondria, confirming this region as a legitimate mitochondrial localization sequence. Despite lower mitochondrial expression of the short isoform, the capacity to deacetylate mitochondrial proteins and to restore mitochondrial respiration is equally robust following transient transfection of either isoform into SIRT3 knockout embryonic fibroblasts. How these alternative transcripts are regulated and whether they modulate distinct targets is unknown. Furthermore, in contrast to exclusive mitochondrial enrichment of endogenous SIRT3, overexpression of both isoforms shows nuclear localization. This overexpression effect, may partially account for previously observed divergent phenotypes attributed to SIRT3.


Social Determinants of Health and Beyond: Information to Help Family Physicians Improve Patient Care.
Social determinants of health (SDOHs) are a theme in this issue. In addition, we include a series of clinical articles to inform family medicine. One helps to demystify the process of obtaining hearing care. Another provides a case report of how a vanishing twin can confuse a newly available test. We also share articles on the early symptoms and signs of femoral insufficiency fractures and a simple test to help diagnose basal cell carcinomas. Family physicians provide their views on point-of-care tests. Positive outcomes are reported for behavioral health integration into family medicine offices and for diabetes education among patients cared for within patient-centered medical homes. A questionnaire can help family physicians identify and facilitate conversations with their patients about adverse childhood experiences.


Semantic detail in the developing verb lexicon: An extension of Naigles and Kako (1993).
Verbs are often uttered before the events they describe. By 2 years of age, toddlers can learn from such an encounter. Hearing a novel verb in transitive sentences (e.g. The boy lorped the cat), even with no visual referent present, they later map it to a causative meaning (e.g. feed) (e.g. Yuan & Fisher, ). How much semantic detail does their verb representation include on this first, underinformative, encounter? Is the representation sparse, including only information for which they have evidence, or do toddlers make more specific guesses about the verb's meaning? In two experiments (N = 76, mean age 27 months), we address this using an event type studied by Naigles and Kako (); they found that when toddlers hear a novel transitive verb while simultaneously viewing a non-causative referent-a contact event such as patting-they map the verb to the contact event. In Experiment 1 we replicated this basic result. Further, toddlers' representations persisted over a 5-minute delay, manifesting again during a retest. In Experiment 2, toddlers heard the verbs while watching two actors converse instead of while seeing contact events. At test, they showed no evidence of mapping the verbs to contact events, either initially or after a 5-minute delay, despite that in prior work they mapped verbs to causative events under identical circumstances. We infer that on hearing a novel verb in a transitive frame, absent a relevant visual scene, toddlers posit a more specific representation than the evidence requires-one that incorporates causative semantics. A video abstract of this article can be viewed at: https://youtu.be/aRCqSTbr6Bw.


Rationale and design of a randomized controlled trial of directly observed hepatitis C treatment delivered in methadone clinics.
Most methadone-maintained injection drug users (IDUs) have been infected with hepatitis C virus (HCV), but few initiate HCV treatment. Physicians may be reluctant to treat HCV in IDUs because of concerns about treatment adherence, psychiatric comorbidity, or ongoing drug use. Optimal HCV management approaches for IDUs remain unknown. We are conducting a randomized controlled trial in a network of nine methadone clinics with onsite HCV care to determine whether modified directly observed therapy (mDOT), compared to treatment as usual (TAU), improves adherence and virologic outcomes among opioid users. We plan to enroll 80 HCV-infected adults initiating care with pegylated interferon alfa-2a (IFN) plus ribavirin, and randomize them to mDOT (directly observed daily ribavirin plus provider-administered weekly IFN) or TAU (self-administered ribavirin plus provider-administered weekly IFN). Our outcome measures are: 1) self-reported and pill count adherence, and 2) end of treatment response (ETR) or sustained viral response (SVR). We will use mixed effects linear models to assess differences in pill count adherence between treatment arms (mDOT v. TAU), and we will assess differences between treatment arms in the proportion of subjects with ETR or SVR with chi square tests. Of the first 40 subjects enrolled: 21 have been randomized to mDOT and 19 to TAU. To date, the sample is 77% Latino, 60% HCV genotype-1, 38% active drug users, and 27% HIV-infected. Our overall retention rate at 24 weeks is 92%, 93% in the mDOT arm and 92% in the TAU arm. This paper describes the design and rationale of a randomized clinical trial comparing modified directly observed HCV therapy delivered in a methadone program to on-site treatment as usual. Our trial will allow rigorous evaluation of the efficacy of directly observed HCV therapy (both pegylated interferon and ribavirin) for improving adherence and clinical outcomes. This detailed description of trial methodology can serve as a template for the development of future DOT programs, and can also guide protocols for studies among HCV-infected drug users receiving methadone for opiate dependence.


Mechanism of antibacterial activity of the white-rot fungus Hypholoma fasciculare colonizing wood.
In a previous study it was shown that the number of wood-inhabiting bacteria was drastically reduced after colonization of beech (Fagus sylvatica) wood blocks by the white-rot fungus Hypholoma fasciculare, or sulfur tuft (Folman et al. 2008). Here we report on the mechanisms of this fungal-induced antibacterial activity. Hypholoma fasciculare was allowed to invade beech and pine (Pinus sylvestris) wood blocks that had been precolonized by microorganisms from forest soil. The changes in the number of bacteria, fungal biomass, and fungal-related wood properties were followed for 23 weeks. Colonization by the fungus resulted in a rapid and large reduction in the number of bacteria (colony-forming units), which was already apparent after 4 weeks of incubation. The reduction in the number of bacteria coincided with fungal-induced acidification in both beech and pine wood blocks. No evidence was found for the involvement of toxic secondary metabolites or reactive oxygen species in the reduction of the number of bacteria. Additional experiments showed that the dominant bacteria present in the wood blocks were not able to grow under the acidic conditions (pH 3.5) created by the fungus. Hence our research pointed at rapid acidification as the major factor causing reduction of wood-inhabiting bacteria upon colonization of wood by H. fasciculare.


The lethal and sub-lethal consequences of entomopathogenic nematode infestation and exposure for adult pine weevils, Hylobius abietis (Coleoptera: Curculionidae).
Entomopathogenic nematodes (EPN) frequently kill their host within 1-2 days, and interest in EPN focuses mainly on their lethality. However, insects may take longer to die, or may fail to die despite being infected, but little is known about the effects of EPN infection on insects, other than death. Here we investigate both lethal and sub-lethal effects of infection by two EPN species, Steinernema carpocapsae and Heterorhabditis downesi, on adults of the large pine weevil, Hylobius abietis. Following 12h nematode-weevil contact in peat, S. carpocapsae killed a significantly higher proportion of weevils (87-93%) than H. downesi (43-57%) at all concentrations tested. Less than 10% of weevils were dead within 2 days, and weevils continued to die for up to 10 days after exposure (LT(50) of 3 days or more). In a separate experiment, live weevils dissected 6 days after a 24h exposure to nematodes on filter paper harbored encapsulated and dead nematodes, showing that weevils could defend themselves against infection. Some live weevils also harbored live nematodes 6 days after they had been removed from the nematode infested medium. Feeding by weevils was not affected by infection with, or exposure to, either species of EPN. We discuss these results in relation to the use of EPN in biological control against H. abietis.


Maternal diet during gestation and lactation modifies the severity of salt-induced hypertension and renal injury in Dahl salt-sensitive rats.
Environmental exposure of parents or early in life may affect disease development in adults. We found that hypertension and renal injury induced by a high-salt diet were substantially attenuated in Dahl SS/JrHsdMcwiCrl (SS/Crl) rats that had been maintained for many generations on the grain-based 5L2F diet compared with SS/JrHsdMcwi rats (SS/Mcw) maintained on the casein-based AIN-76A diet (mean arterial pressure, 116±9 versus 154±25 mm Hg; urinary albumin excretion, 23±12 versus 170±80 mg/d). RNAseq analysis of the renal outer medulla identified 129 and 82 genes responding to a high-salt diet uniquely in SS/Mcw and SS/Crl rats, respectively, along with minor genetic differences between the SS substrains. The 129 genes responding to salt in the SS/Mcw strain included numerous genes with homologs associated with hypertension, cardiovascular disease, or renal disease in human. To narrow the critical window of exposure, we performed embryo-transfer experiments in which single-cell embryos from 1 colony (SS/Mcw or SS/Crl) were transferred to surrogate mothers from the other colony, with parents and surrogate mothers maintained on their respective original diet. All offspring were fed the AIN-76A diet after weaning. Salt-induced hypertension and renal injury were substantially exacerbated in rats developed from SS/Crl embryos transferred to SS/Mcw surrogate mothers. Conversely, salt-induced hypertension and renal injury were significantly attenuated in rats developed from SS/Mcw embryos transferred to SS/Crl surrogate mothers. Together, the data suggest that maternal diet during the gestational-lactational period has substantial effects on the development of salt-induced hypertension and renal injury in adult SS rats.


Positive surgical margins after robotic assisted radical prostatectomy: a multi-institutional study.
Positive surgical margins are an independent predictive factor for biochemical recurrence after radical prostatectomy. We analyzed the incidence of and associative factors for positive surgical margins in a multi-institutional series of 8,418 robotic assisted radical prostatectomies. We analyzed the records of 8,418 patients who underwent robotic assisted radical prostatectomy at 7 institutions. Of the patients 323 had missing data on margin status. Positive surgical margins were categorized into 4 groups, including apex, bladder neck, posterolateral and multifocal. The records of 6,169 patients were available for multivariate analysis. The variables entered into the logistic regression models were age, body mass index, preoperative prostate specific antigen, biopsy Gleason score, prostate weight and pathological stage. A second model was built to identify predictive factors for positive surgical margins in the subset of patients with organ confined disease (pT2). The overall positive surgical margin rate was 15.7% (1,272 of 8,095 patients). The positive surgical margin rate for pT2 and pT3 disease was 9.45% and 37.2%, respectively. On multivariate analysis pathological stage (pT2 vs pT3 OR 4.588, p<0.001) and preoperative prostate specific antigen (4 or less vs greater than 10 ng/ml OR 2.918, p<0.001) were the most important independent predictive factors for positive surgical margins after robotic assisted radical prostatectomy. Increasing prostate weight was associated with a lower risk of positive surgical margins after robotic assisted radical prostatectomy (OR 0.984, p<0.001) and a higher body mass index was associated with a higher risk of positive surgical margins (OR 1.032, p<0.001). For organ confined disease preoperative prostate specific antigen was the most important factor that independently correlated with positive surgical margins (4 or less vs greater than 10 ng/ml OR 3.8, p<0.001). The prostatic apex followed by a posterolateral site was the most common location of positive surgical margins after robotic assisted radical prostatectomy. Factors that correlated with cancer aggressiveness, such as pathological stage and preoperative prostate specific antigen, were the most important factors independently associated with an increased risk of positive surgical margins after robotic assisted radical prostatectomy.