Diet and exercise in the prevention of diabetes.
Individuals with impaired fasting glucose or impaired glucose tolerance are at high risk of progression to type 2 diabetes. Lifestyle modification through change to diet and exercise habit has considerable potential to prevent or delay the onset of this disease. A systematic literature search was undertaken of Medline, EMBASE, the Cochrane library and the Cumulative Index to Nursing and Allied Health Literature for journal articles relevant to the question of whether type 2 diabetes can be prevented by lifestyle change. Four cohort studies in a total of 4864 high risk individuals followed for a period of 2.5-6 years were identified. These showed that lifestyle change may reduce the incidence of type 2 diabetes by 28-59%. Moreover, follow-up studies also indicate that diabetes incidence rates continue to be depressed many years after the discontinuation of a lifestyle intervention. Evidence from a meta-analysis confirms this evidence and suggests that it would be necessary to treat 6.4 (95% confidence interval 5.0-8.4) individuals to prevent or delay one case of diabetes through lifestyle intervention. An examination of weight loss diets (low fat, high protein or Mediterranean) suggests each may be effective but each has limitations requiring care in food selection. Evidence also suggests that the maintenance of weight loss also requires regular exercise with an additional expenditure of approximately 8.4 MJ week(-1) (2000 kcal week(-1)). Diabetes can be prevented by lifestyle change. The challenge is to develop public health approaches to support individuals with respect to incorporating the lifestyle changes needed to reduce the risk of diabetes into their everyday life.


Physiology and pharmacology of erythropoietin.
Human erythropoietin (Epo) is a 30.4 kDa glycoprotein hormone composed of a single 165 amino acid residues chain to which four glycans are attached. The kidneys are the primary sources of Epo, its synthesis is controlled by hypoxia-inducible transcription factors (HIFs). Epo is an essential factor for the viability and proliferation of erythrocytic progenitors. Whether Epo exerts cytoprotection outside the bone marrow still needs to be clarified. Epo deficiency is the primary cause of the anemia in chronic kidney disease (CKD). Treatment with recombinant human Epo (rhEpo, epoetin) can be beneficial not only in CKD but also for other indications, primarily anemia in cancer patients receiving chemotherapy. Considering unwanted events, the administration of rhEpo or its analogs may increase the incidence of thromboembolism. The expiry of the patents for the original epoetins has initiated the production of similar biological medicinal products ('biosimilars'). Furthermore, analogs (darbepoetin alfa, methoxy PEG-epoetin beta) with prolonged survival in circulation have been developed ('biobetter'). New erythropoiesis-stimulating agents are in clinical trials. These include compounds that augment erythropoiesis directly (e.g. Epo mimetic peptides or activin A binding protein) and chemicals that act indirectly by stimulating endogenous Epo synthesis (HIF stabilizers).


Predator diversity and trophic interactions.
The recognition that predators play important roles in ecosystems has prompted research to resolve how combinations of predator species influence ecosystem functions. Interactions among predator species and their prey can lead to a host of linear and nonlinear effects. Understanding the conditions causing these effects is critical for assigning predator species to functional groups in ways that lead to predictive theory of predator diversity effects on trophic interactions. To this end, I provide a synthesis of experiments examining multiple-predator-species effects on mortality of single shared prey. I show how experimental design and experimental venue can determine the conclusion about the importance of predator diversity on trophic interactions. In addition, I link natural history insights on predator species habitat and hunting behavior with linear and nonlinear multiple-predator effects to derive a new concept of predator diversity effects on trophic interactions. This concept holds that the nature of predator diversity effects is contingent upon predator species hunting mode plus predator and prey species habitat domain (defined as the spatial extent to which a microhabitat is used by a species). This concept allows the classification of multiple-predator effects into four broad functional categories: substitutable, nonlinear due to predator species interference, nonlinear due to intraguild predation, and nonlinear due to predator species synergism. Experimental evidence so far provides ample and comparatively equal support for substitutable, interference, and intraguild effects, and equivocal support for nonlinear synergisms. The paper closes by discussing ways to further a research program aimed at using the building blocks presented here to understand predator functional diversity and trophic interactions in complex ecological systems.


Socioeconomic status inconsistency and risk of stroke among Japanese middle-aged women.
Little research has been conducted to examine the effect of inconsistencies in socioeconomic status on cardiovascular health. In particular, no studies have been reported in Asian countries, including Japan, which is thought to have high socioeconomic status inconsistency among women. We examined the effect of status inconsistency between education level and occupation on stroke risk in a prospective 20-year study of 14 742 middle-aged Japanese women included in the prospective Japan Public Health Center-based (JPHC) Study Cohort I in 1990. Status inconsistency between education level and occupation was determined (qualified, overqualified, and underqualified), and the association with risk of stroke was examined. Cox proportional regression analysis was used to determine hazard ratios, which were adjusted for age, marital status, and geographical area. Adjusted hazard ratio for stroke in overqualified compared with qualified women was 2.06 (95% confidence interval, 1.13-3.78). Adjusted hazard ratios for stroke among highly educated manual workers and workers in service industry were 3.47 (95% confidence interval, 1.54-7.84) and 3.21 (95% confidence interval, 1.49-6.90), respectively, when compared with highly educated professionals/managers. High academic qualifications without an appropriate job could be a risk factor for stroke among Japanese women. Our result suggests that status inconsistency could be a potential explanation for the increased stroke risk among highly educated women.


Pax7 is necessary and sufficient for the myogenic specification of CD45+:Sca1+ stem cells from injured muscle.
CD45(+):Sca1(+) adult stem cells isolated from uninjured muscle do not display any myogenic potential, whereas those isolated from regenerating muscle give rise to myoblasts expressing the paired-box transcription factor Pax7 and the bHLH factors Myf5 and MyoD. By contrast, CD45(+):Sca1(+) isolated from injured Pax7( -/-) muscle were incapable of forming myoblasts. Infection of CD45(+):Sca1(+) cells from uninjured muscle with retrovirus expressing Pax7 efficiently activated the myogenic program. The resulting myoblasts expressed Myf5 and MyoD and differentiated into myotubes that expressed myogenin and myosin heavy chain. Infection of CD45(-):Sca1(-) cells from Pax7( -/-) muscle similarly gave rise to myoblasts. Notably, infection of Pax7-deficient muscle with adenoviral Pax7 resulted in the de novo formation of regenerated myofibers. Taken together, these results indicate that Pax7 is necessary and sufficient to induce the myogenic specification of CD45(+) stem cells resident in adult skeletal muscle. Moreover, these experiments suggest that viral transduction of Pax7 is a potential therapeutic approach for the treatment of neuromuscular degenerative diseases.


Neuroimaging in cannabis use: a systematic review of the literature.
We conducted a systematic review to assess the evidence for specific effects of cannabis on brain structure and function. The review focuses on the cognitive changes associated with acute and chronic use of the drug. We reviewed literature reporting neuroimaging studies of chronic or acute cannabis use published up until January 2009. The search was conducted using Medline, EMBASE, LILACS and PsycLIT indexing services using the following key words: cannabis, marijuana, delta-9-tetrahydrocannabinol, THC, cannabidiol, CBD, neuroimaging, brain imaging, computerized tomography, CT, magnetic resonance, MRI, single photon emission tomography, SPECT, functional magnetic resonance, fMRI, positron emission tomography, PET, diffusion tensor MRI, DTI-MRI, MRS and spectroscopy. Sixty-six studies were identified, of which 41 met the inclusion criteria. Thirty-three were functional (SPECT/PET/fMRI) and eight structural (volumetric/DTI) imaging studies. The high degree of heterogeneity across studies precluded a meta-analysis. The functional studies suggest that resting global and prefrontal blood flow are lower in cannabis users than in controls. The results from the activation studies using a cognitive task are inconsistent because of the heterogeneity of the methods used. Studies of acute administration of THC or marijuana report increased resting activity and activation of the frontal and anterior cingulate cortex during cognitive tasks. Only three of the structural imaging studies found differences between users and controls. Functional neuroimaging studies suggest a modulation of global and prefrontal metabolism both during the resting state and after the administration of THC/marijuana cigarettes. Minimal evidence of major effects of cannabis on brain structure has been reported.


The failure of a major histocompatibility antigen to stimulate a thyroid allograft reaction after culture in oxygen.
The lymphocytic infiltration found in multiple cultured BALB/c thyroids placed in the same C57BL/6 recipient was found to be highly correlated and the high variability between animals was not influenced by the number of lobes. It was concluded that the variable infiltration was largely due to host factors. Because a similar correlation was found between BALB/c and C3H grafts, the response to a minor antigen common to these two strains was suggested as a cause of the infiltration. When the response of C57BL/6 mice to cultured B6.C (H-2d) and BALB.B (H-2b) grafts was compared, a synergism between major and minor antigens was suggested. However, when the time of culture was increased from 48 to 60 h and the response of BALB/c and C57BL/6 mice were compared with identically cultured B6.C (H-2d) grafts, a striking difference between major and minor antigens was observed. None of 10 such grafts in C57BL/6 recipients (major antigens only) showed any infiltration, whereas 8 out of the 9 grafts in BALB/c recipients (minor antigens only) were infiltrated.


"Not on the agenda": A qualitative study of influences on health services use among poor young women who use drugs in Cape Town, South Africa.
Poor young women who use alcohol and other drugs (AODs) in Cape Town, South Africa, need access to health services to prevent HIV. Efforts to link young women to services are hampered by limited information on what influences service initiation. We explored perceptions of factors that influence poor AOD-using young women's use of health services. We conducted four focus groups with young women (aged 16-21) who used AODs and were recruited from two township communities in Cape Town. We also conducted 14 in-depth interviews with health and social welfare service planners and providers. Discussion topics included young women's use of health services and perceived influences on service use. Qualitative data were analysed using a framework approach. The findings highlighted structural, contextual, and systemic influences on the use of health services by young women who use AODs. First, young women were absent from the health agenda, which had an impact on the provision of women-specific services. Resource constraints and gender inequality were thought to contribute to this absence. Second, gender inequality and stigma toward young women who used AODs led to their social exclusion from education and employment opportunities and health care. Third, community poverty resulted in the emergence of perverse social capital and social disorder that limited social support for treatment. Fourth, the health care system was unresponsive to the multiple service needs of these young women. To reach young women who use AODs, interventions need to take cognisance of young women's risk environment and health systems need to adapt to respond better to their needs. For these interventions to be effective, gender must be placed on the policy agenda.


[Women's satisfaction with waiting times for further investigation in breast cancer screening].
To determine the factors associated with satisfaction with waiting times for further investigation in breast cancer screening. We carried out a cross-sectional study by telephone survey of a representative sample of women (N=316) participating in the breast cancer screening program of the autonomous region of Valencia (Spain) who required additional tests to confirm the diagnosis. Descriptive analysis was performed by contingency tables (p<0.05) and multivariate association by odds ratios (OR) of logistic regression models (95%CI). Satisfaction with the waiting time was 78.6%. A higher risk of dissatisfaction was found in women from a "high" social class (OR=3.17; 95% CI: 1.10-9.14), those who perceived that the waiting time was "more than 2 weeks", both "since the notification of the need for further investigation until completion of the first test" (OR=15,54; 95%CI: 5,87-41,12) and "since the completion of the last test until notification of the final result" (OR=11.57; 95% CI: 2.96-45.19), and in women who experienced the attention as "worse than expected" (OR=15.40; 95% CI: 1.41-168.64). The maximum waiting time acceptable to the highest percentage of women was "up to 1 week" for each waiting period (n=47, 73.5%; n=14, 45.2%). Waiting times of no more than 1 week and never more than 2 weeks for each waiting period are recommended. Women should be given an approximate waiting time, paying special attention to women aged 45 to 54 years attending their initial screening.


Synthetic biology approaches to engineering the nitrogen symbiosis in cereals.
Nitrogen is abundant in the earth's atmosphere but, unlike carbon, cannot be directly assimilated by plants. The limitation this places on plant productivity has been circumvented in contemporary agriculture through the production and application of chemical fertilizers. The chemical reduction of nitrogen for this purpose consumes large amounts of energy and the reactive nitrogen released into the environment as a result of fertilizer application leads to greenhouse gas emissions, as well as widespread eutrophication of aquatic ecosystems. The environmental impacts are intensified by injudicious use of fertilizers in many parts of the world. Simultaneously, limitations in the production and supply of chemical fertilizers in other regions are leading to low agricultural productivity and malnutrition. Nitrogen can be directly fixed from the atmosphere by some bacteria and Archaea, which possess the enzyme nitrogenase. Some plant species, most notably legumes, have evolved close symbiotic associations with nitrogen-fixing bacteria. Engineering cereal crops with the capability to fix their own nitrogen could one day address the problems created by the over- and under-use of nitrogen fertilizers in agriculture. This could be achieved either by expression of a functional nitrogenase enzyme in the cells of the cereal crop or through transferring the capability to form a symbiotic association with nitrogen-fixing bacteria. While potentially transformative, these biotechnological approaches are challenging; however, with recent advances in synthetic biology they are viable long-term goals. This review discusses the possibility of these biotechnological solutions to the nitrogen problem, focusing on engineering the nitrogen symbiosis in cereals.


Elevated plasma glucose-dependent insulinotropic polypeptide associates with hyperinsulinemia in impaired glucose tolerance.
The role of gut-derived incretin, glucose-dependent insulinotropic polypeptide (also known as gastric inhibitory peptide [GIP]), in compensatory beta-cell hypersecretion during insulin-resistant states and in transition to beta-cell failure in type 2 diabetes is unknown. We carried out oral glucose tolerance testing followed by blood sampling 10 times for 2 h on 68 age- and BMI-matched participants of the Baltimore Longitudinal Study on Aging (BLSA) with normal glucose tolerance (34 subjects), impaired glucose tolerance (IGT) (18 subjects with both impaired fasting and 2-h plasma glucose levels), and type 2 diabetes (16 subjects with both diabetic fasting and 2-h plasma glucose levels). We assayed plasma glucose, insulin, C-peptide, glucagon, and intact and total GIP levels and quantitated glucose and hormone responses to the oral glucose tolerance test. We also compared GIP and insulin release and sensitivity indexes between groups. After glucose ingestion, subjects with IGT had both hyperinsulinemia and hyperemia, while subjects with type 2 diabetes had both beta- and GIP-cell deficiency. In the former group, there was also a significant positive correlation between the augmented plasma intact and total GIP levels and both fasting and post-oral glucose load plasma insulin levels. Elevated plasma GIP levels are correlated with hyperinsulinemia in the impaired glucose-tolerant state, whereas type 2 diabetes is associated with a failure to secrete adequate amounts of both GIP and insulin, indicating a common pathway of resistance to and eventually failure of glucose responsiveness in beta- and GIP-cells.


CDK9 activity is critical for maintaining MDM4 overexpression in tumor cells.
The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.


Proposing a causal pathway for health-related quality of life in people with psychotic disorders.
This study aimed to establish independent predictors for health-related quality of life (HRQoL) in people with psychotic disorders, and the mediating role of global functioning in those relationships. Data for 1642 people collected as part of the second Australian National Survey of Psychosis were analyzed. The Assessment of Quality of Life (AQoL)-4D and the Personal and Social Performance scale were used for assessing HRQoL and global functioning respectively. The study commenced with a theoretical model comprised of 26 sociodemographic and clinical variables. A predictive model for HRQoL was built up using a purposeful selection strategy to arrive at a set of clinically meaningful, independent predictors. The mediating effect of global functioning was then assessed. Seven variables were found to have an independent effect on HRQoL: perception of loneliness, number of negative symptoms, use of psychotropic and anticholinergic medications, course of disorder, lifetime histories of chronic pain and cardiovascular disease and living arrangements at the time of the interview. All variables except perceived loneliness and chronic pain were partially mediated through global functioning. This final model explained 46% of the variance in HRQoL, with loneliness and number of negative symptoms the strongest predictors. Evidence in support of a credible causal pathway for HRQoL in people with psychotic disorders, mediated by global functioning was presented. The importance of the quality of social relationships was highlighted, and potential targets for improving the HRQoL of this population identified.


External memory aids for memory problems in people with multiple sclerosis: A systematic review.
Approximately 40-60% of people with multiple sclerosis (MS) have memory problems, which adversely impact on their everyday functioning. Evidence supports the use of external memory aids in people with stroke and brain injury, and suggests they may reduce everyday memory problems in people with MS. Previous reviews of people with MS have only evaluated randomised trials; therefore this review included other methodologies. The aim was to assess the efficacy of external memory aids for people with MS for improving memory functioning, mood, quality of life, and coping strategies. Seven databases were systematically searched. Intervention studies that involved training in the use of external memory aids, e.g., personal digital assistants, with at least 75% of people with MS, were included. Based on study design, quality was rated with the SCED or PEDro scale. Nine studies involving 540 participants were included. One single case experimental design (mean of 8 on SCED scale) and eight group studies (mean of 5 on PEDro scale) were included. One study reported a significant treatment effect on subjective memory functioning, two on mood, and two on coping strategies. There is insufficient evidence to support or refute the effectiveness of external memory aids for improving memory function in people with MS.


Hypokalemia in children with severe falciparum malaria.
Acidosis is now recognized as an important component of the severe malaria syndrome and a predictor of fatal outcome. Alterations in plasma potassium concentrations are commonly associated with acidosis. To date, there is little information about the changes in potassium in severe malaria. Prospective study examining the changes in plasma potassium in the first 24 hrs following admission in children with severe malaria. Urinary fractional excretion of potassium and the transtubular gradient of potassium were examined at admission. High-dependency unit on the coast of Kenya. Kenyan children admitted to hospital with clinical features of severe malaria (impaired consciousness or deep breathing) complicated by acidosis (base deficit >8). Children received standard therapy for severe malaria; in addition, they received boluses of either 0.9% saline or 4.5% human albumin solution to correct hypovolemia, and intravenous potassium replacement was prescribed to children who developed hypokalemia (plasma potassium <3 mmol/L). Thirty-eight Kenyan children were recruited with severe malaria and acidosis. At admission, serum potassium was normal (3-5.5 mmol/L) in 31 (81.6%) and low (<3 mmol/L) in four (11%) children, and three (6.3%) children had hyperkalemia (>5.5 mmol/L). Plasma potassium decreased rapidly within 4-8 hrs of admission: 15 (40%) patients were hypokalemic (<3 mmol/L); of these, five (13%) had plasma potassium of <2.5 mmol/L. Fractional excretion of potassium and the transtubular gradient of potassium were above normal range, indicating renal potassium loss. Hypokalemia is a common complication of severe malaria; however, it is often not apparent on admission. On correction of acidosis, plasma potassium decreases precipitously, and thus careful, serial monitoring of serum potassium is suggested in patients with severe malaria complicated by acidosis.


The influence of menopause status and postmenopausal use of hormone therapy on presentation of major depression in women.
The purpose of this study was to determine whether there are differences in depression characteristics among premenopausal, perimenopausal, and postmenopausal women with major depressive disorder. This study also evaluated these differences between postmenopausal women with major depressive disorder who are taking and not taking hormone therapy. Analyses conducted with data from the Sequenced Treatment Alternatives to Relieve Depression study focused on female outpatients with nonpsychotic major depressive disorder seeking treatment in 41 primary or psychiatric care settings across the United States. Baseline demographic and clinical characteristics were compared among women not taking hormone therapy who were premenopausal (n = 950), perimenopausal (n = 380), or postmenopausal (n = 562). These comparisons were also made between postmenopausal women (n = 768) taking (n = 171) or not taking (n = 562) hormone therapy. After adjusting for sociodemographic and clinical baseline differences, premenopausal women were more likely to present with irritability than were either perimenopausal or postmenopausal women and were more likely to have decreased appetite and less likely to have early-morning insomnia than were perimenopausal women. Postmenopausal women were more likely to have suicidal ideation and poorer physical functioning than were either of the other groups and were more likely to have sympathetic arousal and gastrointestinal symptoms than were premenopausal women. After adjusting for baseline differences, postmenopausal women taking hormone therapy had better physical functioning, fewer melancholic features, less sympathetic arousal, and more lack of involvement in activities than did women not taking hormone therapy. Menopause status and postmenopausal use of hormone therapy may influence the clinical presentation of major depressive episodes in women.


Quantitative proteomics analysis of BMS-214662 effects on CD34 positive cells from chronic myeloid leukaemia patients.
Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell and is driven by the Bcr-Abl oncoprotein. Abl kinase inhibitors (protein tyrosine kinase inhibitors) represent standard treatment for CML and induce remission in the majority of patients with early disease, however these drugs do not target leukaemic stem cells (LSCs) effectively, thus preventing cure. Previously, we identified the farnesyl transferase inhibitor BMS-214662 as a selective inducer of apoptosis in LSCs of CML patients relative to normal controls; however, the mechanism underlying LSC-specific apoptosis remains unclear. To identify pathways involved in the favourable effects of BMS-214662 in CML, we employed a proteomic approach (based on iTRAQ) to analyse changes in protein expression in response to drug treatment in the nuclear and cytoplasmic fractions of CD34(+) CML cells. The study identified 88 proteins as altered after drug treatment, which included proteins known to be involved in nucleic acid metabolism, oncogenesis, developmental processes and intracellular protein trafficking. We found that expression of Ebp1, a negative regulator of proliferation, was upregulated in the nucleus of BMS-214662-treated cells. Furthermore, proteins showing altered levels in the cytosol, such as histones, were predominantly derived from the nucleus and BMS-214662 affected expression levels of nuclear pore complex proteins. Validation of key facets of these observations suggests that drug-induced alterations in protein localisation, potentially via loss of nuclear membrane integrity, contributes to the LSC specificity of BMS-214662, possibly via Ran proteins as targets.


Intratumoral Delivery of IL-21 Overcomes Anti-Her2/Neu Resistance through Shifting Tumor-Associated Macrophages from M2 to M1 Phenotype.
Tumor resistance is a major hurdle to anti-Her2/neu Ab-based cancer therapy. Current strategies to overcome tumor resistance focus on tumor cell-intrinsic resistance. However, the extrinsic mechanisms, especially the tumor microenvironment, also play important roles in modulating the therapeutic response and resistance of the Ab. In this study, we demonstrate that tumor progression is highly associated with TAMs with immune-suppressive M2 phenotypes, and deletion of TAMs markedly enhanced the therapeutic effects of anti-Her2/neu Ab in a HER2/neu-dependent breast cancer cell TUBO model. Tumor local delivery of IL-21 can skew TAM polarization away from the M2 phenotype to a tumor-inhibiting M1 phenotype, which rapidly stimulates T cell responses against tumor and dramatically promotes the therapeutic effect of anti-Her2 Ab. Skewing of TAM polarization by IL-21 relies substantially on direct action of IL-21 on TAMs rather than stimulation of T and NK cells. Thus, our findings identify the abundant TAMs as a major extrinsic barrier for anti-Her2/neu Ab therapy and present a novel approach to combat this extrinsic resistance by tumor local delivery of IL-21 to skew TAM polarization. This study offers a therapeutic strategy to modulate the tumor microenvironment to overcome tumor-extrinsic resistance.


The reliability and predictive validity of the Guidelines for Stalking Assessment and Management (SAM).
This study assessed the reliability and validity of the Guidelines for Stalking Assessment and Management (SAM), a structured professional judgment measure for assessing stalking risks. The SAM was completed retrospectively from file review for 146 adult stalkers (90.4% male) referred to a community-based forensic mental health service. Interrater reliability (IRR) was initially poor, but developing a strict definition of stalking currency and rescoring the SAM led to improvement. Based on the updated scoring, IRR was moderate for judgments about whether stalking was ongoing at the time of assessment, and fair to moderate for summary risk judgments. Both case prioritization (area under the curve [AUC] = .69) and risk for continued stalking (AUC = .76) ratings discriminated between groups, with high-risk stalkers 5-9 times as likely as low-risk stalkers to reoffend by stalking their original victims. Lifetime SAM total scores (AUC = .70) also featured moderate to good discrimination. Follow-up analyses suggested that this was driven mainly by the recent presence of risk markers and the nature of any ongoing stalking situation rather than historical or individual factors. Findings support the use of the SAM to structure risk judgments made by those with experience in assessing stalking. Current results also imply that IRR might be improved by introducing (a) a fixed definition of stalking currency and (b) usage guidelines for specific contexts. (PsycINFO Database Record (c) 2018 APA, all rights reserved).


Conjugated linolenic acid (CLnA), conjugated linoleic acid (CLA) and other biohydrogenation intermediates in plasma and milk fat of cows fed raw or extruded linseed.
Thirty lactating dairy cows were used in a 3 × 3 Latin-square design to investigate the effects of a raw or extruded blend of linseed and wheat bran (70:30) on plasma and milk fatty-acids (FA). Linseed diets, containing 16.6% linseed blend on a dry-matter basis, decreased milk yield and protein percentage. They decreased the proportions of FA with less than 18 carbons in plasma and milk and resulted in cis-9, cis-12, cis-15 18:3 proportions that were more than three and four times higher in plasma and milk, respectively, whereas cis-9, cis-12 18:2 proportions were decreased by 10-15%. The cis-9, trans-11, cis-15 18:3 isomer of conjugated linolenic acid was not detected in the milk of control cows, but was over 0.15% of total FA in the milk fat of linseed-supplemented cows. Similarly, linseed increased plasma and milk proportions of all biohydrogenation (BH) intermediates in plasma and milk, including the main isomer of conjugated linoleic acid cis-9, trans-11 18:2, except trans-4 18:1 and cis-11, trans-15 18:2 in plasma lipids. In milk fat, compared with raw linseed, extruded linseed further reduced 6:0-16:0 even-chain FA, did not significantly affect the proportions of 18:0, cis-9 18:1 and cis-9, cis-12 18:2, tended to increase cis-9, cis-12, cis-15 18:3, and resulted in an additional increase in the proportions of most BH intermediates. It was concluded that linseed addition can improve the proportion of conjugated linoleic and linolenic acids, and that extrusion further increases the proportions of intermediates of ruminal BH in milk fat.


DEPA classification: a proposal for standardising PRP use and a retrospective application of available devices.
Significant biological differences in platelet-rich plasma (PRP) preparations have been highlighted and could explain the large variability in the clinical benefit of PRP reported in the literature. The scientific community now recommends the use of classification for PRP injection; however, these classifications are focused on platelet and leucocyte concentrations. This presents the disadvantages of (1) not taking into account the final volume of the preparation; (2) omitting the presence of red blood cells in PRP and (3) not assessing the efficiency of production. On the basis of standards classically used in the Cell Therapy field, we propose the DEPA (Dose of injected platelets, Efficiency of production, Purity of the PRP, Activation of the PRP) classification to extend the characterisation of the injected PRP preparation. We retrospectively applied this classification on 20 PRP preparations for which biological characteristics were available in the literature. Dose of injected platelets varies from 0.21 to 5.43 billion, corresponding to a 25-fold increase. Only a Magellan device was able to obtain an A score for this parameter. Assessments of the efficiency of production reveal that no device is able to recover more than 90% of platelets from the blood. Purity of the preparation reveals that a majority of the preparations are contaminated by red blood cells as only three devices reach an A score for this parameter, corresponding to a percentage of platelets compared with red blood cells and leucocytes over 90%. These findings should provide significant help to clinicians in selecting a system that meets their specific needs for a given indication.


Managing soft tissue sarcomas in a developing country: are prognostic factors similar to those of developed world?
Managing soft tissue sarcomas (STS) in a developing country with limited financial resources and a poor health referral system is a challenge. Presenting late, these extremity STS are prone to recurrence despite apparently complete resection. This study aimed to explore and compare the impact of clinico-pathological factors on recurrence and survival in Pakistan with the corresponding figures quoted from the developed world. An institutional review was performed on all patients with primary STS of the extremities operated on between 1994 and 2008. The prognostic influence of clinical, pathologic, and treatment variables on local recurrence free survival (LRFS), metastasis free survival (MFS) and overall survival (OS) were analyzed by univariate and multivariate Cox regression analysis and Kaplan Meier survival curves. A total of 84 patients with a mean age of 41.8 ± 21.9 years were included in the study. The local recurrence rate was 14.3% after a median of 6 (mean 7.4) months. Metastases occurred in 7 patients (8.3%) and 65 patients were alive without evidence of disease after a mean follow-up of 52.6 ± 39.8 months. Tumor size > 5 cm, grade 3 tumors and margin < 10 mm significantly increased local recurrence rates. A margin ≥ 10 mm and age < 45 years significantly enhanced cumulative survival. Significant multivariate risk factors for metastases were margin < 10 mm and tumor grade G3. Despite a poor health referral system in our country, our results are no different from those reported from the developed world. Surgical margins and tumor grade prognostically influenced LRFS, MFS and OS.


Associations between HIV-related stigma, self-esteem, social support, and depressive symptoms in Namibia.
The current study sought to investigate the association between HIV-related stigma, self-esteem, social support, and depression of people living with HIV and AIDS (PLWHA) in Namibia. Purposive sampling was used to recruit a total of 124 men and women living with HIV/AIDS in the Katima Mulilo region of northern Namibia. A cross-sectional survey was used to collect information on demographics, self-esteem, social support, HIV-related stigma, and depression. Correlation analysis revealed that HIV-related stigma, self-esteem, and social support were all significantly correlated with depression. Further, Ordinary Least Squares (OLS) regression analysis indicated that HIV-related stigma was the largest risk factor and self-esteem was the largest protective factor with respect to depressive symptoms. Findings indicated the necessity of appropriate assessment and intervention for psychosocial distress among PLWHA. Helping professionals should design evidence-based interventions that address individual and societal challenges that impact people living with HIV and AIDS.


Monitoring antigen-specific biologics: current knowledge and future prospects.
An increasing number of antigen-specific biologics have been introduced into clinical practice, and the ones that arrived first have already reached the end of their patented life span. Despite the use of these agents for over a decade, individualized dosing is not standard practice. Most patients are treated according to treatment protocols, with a fixed dose administered at fixed time intervals. Although the between-subject variability in the volume of distribution is small, there is a moderate to high between-subject variability in the clearance of these biologics. The formation of neutralizing antidrug antibodies (ADAs) further contributes to the variability in the pharmacokinetics and pharmacodynamics. After the development of assays to detect biologic drug serum concentrations and ADA titers, the first clinical studies in immune-mediated diseases such as rheumatology, gastroenterology, and dermatology have now shown clear concentration-effect relationships. By monitoring the serum trough concentrations of biologics, patients with high drug exposure could be identified and dose reductions in those patients may lead to improved safety and substantial cost savings. Low biologic drug serum concentrations may be due to the development of ADAs, and if these are detected, a switch to an alternative treatment is indicated. We envision a vast expansion of therapeutic drug monitoring to support the use of biologics, and we urge the International Association of Therapeutic Drug Monitoring and Clinical Toxicology to embark on initiatives to investigate the contribution of therapeutic drug monitoring to this field.


Repression of prespliceosome complex formation at two distinct steps by Fox-1/Fox-2 proteins.
Precise and robust regulation of alternative splicing provides cells with an essential means of gene expression control. However, the mechanisms that ensure the tight control of tissue-specific alternative splicing are not well understood. It has been demonstrated that robust regulation often results from the contributions of multiple factors to one particular splicing pathway. We report here a novel strategy used by a single splicing regulator that blocks the formation of two distinct prespliceosome complexes to achieve efficient regulation. Fox-1/Fox-2 proteins, potent regulators of alternative splicing in the heart, skeletal muscle, and brain, repress calcitonin-specific splicing of the calcitonin/CGRP pre-mRNA. Using biochemical analysis, we found that Fox-1/Fox-2 proteins block prespliceosome complex formation at two distinct steps through binding to two functionally important UGCAUG elements. First, Fox-1/Fox-2 proteins bind to the intronic site to inhibit SF1-dependent E' complex formation. Second, these proteins bind to the exonic site to block the transition of E' complex that escaped the control of the intronic site to E complex. These studies provide evidence for the first example of regulated E' complex formation. The two-step repression of presplicing complexes by a single regulator provides a powerful and accurate regulatory strategy.


Solvent influence on the thermodynamics for hydride transfer from bis(diphosphine) complexes of nickel.
The thermodynamic hydricity of a metal hydride can vary considerably between solvents. This parameter can be used to determine the favourability of a hydride-transfer reaction, such as the reaction between a metal hydride and CO2 to produce formate. Because the hydricities of these species do not vary consistently between solvents, reactions that are thermodynamically unfavourable in one solvent can be favourable in others. The hydricity of a water-soluble, bis-phosphine nickel hydride complex was compared to the hydricity of formate in water and in acetonitrile. Formate is a better hydride donor than [HNi(dmpe)2](+) by 7 kcal mol(-1) in acetonitrile, and no hydride transfer from [HNi(dmpe)2](+) to CO2 occurs in this solvent. The hydricity of [HNi(dmpe)2](+) is greatly improved in water relative to acetonitrile, in that reduction of CO2 to formate by [HNi(dmpe)2](+) was found to be thermodynamically downhill by 8 kcal mol(-1). Catalysis for the hydrogenation of CO2 was pursued, but the regeneration of [HNi(dmpe)2] under catalytic conditions was unfavourable. However, the present results demonstrate that the solvent dependence of thermodynamic parameters such as hydricity and acidity can be exploited in order to produce systems with balanced or favourable overall thermodynamics. This approach should be advantageous for the design of future water-soluble catalysts.


Phenotypic characterization of Escherichia coli through whole-cell fatty acid profiling to investigate host specificity.
The objective of the study was to investigate whole-cell fatty acid methyl ester (FAME) profiles of 605 Escherichia coli isolates to determine their host specificity. The isolates were cultured from six possible sources of fecal pollution; 180 isolates from sewage, 85 from dairy cow, 98 from chicken, 76 from swine, 94 from deer, and 72 from waterfowl, mostly geese and ducks. The FAME profiles were presented as the relative masses of 12 FAMEs identified in the isolates and it was found that none of the six hosts carried a "signature" FAME, a FAME that is uniquely associated with a particular host category. However, two-sample t-test analyses indicated that the mean relative masses of seven FAMEs out of the 12 identified showed statistically significant differences (95% confidence interval) between isolates of human and non-human origins. In addition, a linear discriminant function based on mean relative mass variations in individual FAMEs classified the known-source isolates into their respective host categories with a 47.6% average rate of correct classification (ARCC) in a six-way discriminant analysis. The ARCC increased to 61.3% when the individual hosts were pooled into larger categories of human, livestock, and wildlife. The accuracy was 75.5% when isolates of human origin were discriminated against those of non-human origins. Random cluster formation analysis indicated that the library size was sufficient to prevent random grouping among the isolates.


Comparing algorithms for automated vessel segmentation in computed tomography scans of the lung: the VESSEL12 study.
The VESSEL12 (VESsel SEgmentation in the Lung) challenge objectively compares the performance of different algorithms to identify vessels in thoracic computed tomography (CT) scans. Vessel segmentation is fundamental in computer aided processing of data generated by 3D imaging modalities. As manual vessel segmentation is prohibitively time consuming, any real world application requires some form of automation. Several approaches exist for automated vessel segmentation, but judging their relative merits is difficult due to a lack of standardized evaluation. We present an annotated reference dataset containing 20 CT scans and propose nine categories to perform a comprehensive evaluation of vessel segmentation algorithms from both academia and industry. Twenty algorithms participated in the VESSEL12 challenge, held at International Symposium on Biomedical Imaging (ISBI) 2012. All results have been published at the VESSEL12 website http://vessel12.grand-challenge.org. The challenge remains ongoing and open to new participants. Our three contributions are: (1) an annotated reference dataset available online for evaluation of new algorithms; (2) a quantitative scoring system for objective comparison of algorithms; and (3) performance analysis of the strengths and weaknesses of the various vessel segmentation methods in the presence of various lung diseases.


Mutation of Rv2887, a marR-like gene, confers Mycobacterium tuberculosis resistance to an imidazopyridine-based agent.
Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation.


Online Sexual Experiences Predict Subsequent Sexual Health and Victimization Outcomes Among Female Adolescents: A Latent Class Analysis.
Adolescents' online sexual experiences (e.g., pornography use, sexual chatting, sexualized social media use, and nude image exchange) provide a new context for sexual socialization. Traditionally, online sexual experiences are often aggregated averages, which neglect their complexity and fail to identify individual differences in the experience. Moreover, the lack of longitudinal research in this area has failed to determine if these experiences predict later offline sexual health and violence outcomes. An analysis of two waves of surveys completed by ethnically and socioeconomically diverse female adolescents (N = 296; 49% maltreated; aged 14-16 years) participating in a larger cross-sequential study was conducted to address these gaps. Established latent classes from the prerequisite study of online sexual experiences at Time 1 were Online Abstinent (low probability of any online sexual experiences), Online Inclusive (high probability of all online sexual experiences), Attractors (high probability of attracting attention from others), and Seekers (high probability of seeking out sexual content and interaction). Class membership uniquely predicted HIV risk, number of physically violent romantic partners, and the occurrence of sexual assault at Time 2. Although membership in risker online sexual experience classes predicted later offline risk and victimization, this was especially true for maltreated participants. These findings demonstrate the advantages of examining online sexual experiences in a way that emphasizes their complexity and individual differences in influential susceptibility.


The structural and optical properties of type III human collagen biosynthetic corneal substitutes.
The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2-9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants.


Characterization of the transferrin-binding protein in a human trophoblast.
The physical properties and binding characteristics of the solubilized transferrin-binding protein from BeWo cells, a human choriocarcinoma cell line, were investigated. The binding protein was isolated from 125I-labelled membranes by solubilization followed by immunoprecipitation with anti-human transferrin in the presence of saturating human transferrin. Gel filtration on acrylamide agarose (AcA-22) at 21 degrees C in the absence of transferrin indicates that the transferrin-binding protein has a Stokes' radius of 4.6 nm. In the presence of transferrin, the Stokes' radius of the transferrin-binding BeWo protein increases to 6.3 nm. Parallel sucrose density centrifugation studies indicate that the BeWo protein has a sedimentation coefficient of 9.4 S in the absence of transferrin and 10.9 S in the presence of transferrin. Relative molecular mass calculations from sedimentation studies in H2O and D2O, using the method of Sadler et al (1979), indicate a relative molecular mass of 204,000 for the solubilized receptor and 354,000 for the receptor in the presence of transferrin.


Puerperal symphysis fundus distance: normal values.
To measure puerperal symphysis fundus distance (SFD) by two manual methods and to calculate individual normal values. Prospective cohort study including women after single pregnancy without special problems in uterine involution. SFD was daily measured on the 1(st)-5(th) postpartum days by counting the number of midwife's fingers put horizontally on the mother's abdomen and by a paper tape, respectively. Linear mixed model analysis was performed to determine SFD normal values. Independent variables determining the SFD on different days after delivery were the gestational age and the delivery mode (finger method) and the maternal age, body mass index (BMI), delivery mode and birth weight (tape method). Individual normal values were calculated by regression equation including the coefficients for the independent variables. Puerperal SFD values measured by tape have more independent variables than those measured by fingers and reflect better the actual situation of the patient. It should be verified whether values deviating from normal values of tape measurements may prevent postpartum complications.


Clusters of DNA double-strand breaks induced by different doses of nitrogen ions for various LETs: experimental measurements and theoretical analyses.
The yields and clustering of DNA double-strand breaks (DSBs) were investigated in normal human skin fibroblasts exposed to gamma rays or to a wide range of doses of nitrogen ions with various linear energy transfers (LETs). Data obtained by pulsed-field gel electrophoresis on the dose and LET dependence of DNA fragmentation were analyzed with the randomly located clusters (RLC) formalism. The formalism considers stochastic clustering of DSBs along a chromosome due to chromatin structure, particle track structure, and multitrack action. The relative biological effectiveness (RBE) for the total DSB yield did not depend strongly on LET, but particles with higher LET produced higher fractions of small DNA fragments, corresponding in the formalism to an increase in the average number of DSBs per DSB cluster. The results are consistent with the idea that DSB clustering along chromosomes is what leads to large RBEs of high-LET radiations for major biological end points. At a given dose, large fragments are less affected by the variability in LET than small fragments, suggesting that the two free ends in large fragments are often produced by two different tracks. The formalism successfully described an extra increase in small DNA fragments as dose increases and a related decrease in large fragments, mainly due to interlacing of DSB clusters produced along a chromosome by different tracks, since interlacing cuts larger DNA fragments into smaller ones.


Determinants of sensitization of carotid baroreceptors by pulsatile pressure in dogs.
The threshold pressure of single baroreceptor units is decreased after compared with before exposure to pulsatile pressure according to previous studies in our laboratory. The purpose of the present study is to characterize the determinants of sensitization of arterial baroreceptors by pulsatile pressure. Carotid sinus nerve activity was recorded in dogs anesthetized with chloralose. Two indexes of baroreceptor "sensitivity" were obtained by comparing nerve activity before and immediately after exposure of the isolated carotid sinus to pulsatile pressure for periods up to 10 minutes. Sensitization occurred 1) when the threshold pressure of single baroreceptor units determined with a slow nonpulsatile ramp decreased after as compared with before pulsing and 2) when multiple unit activity increased after as compared with before pulsing at various mean levels of static pressure. Sensitization was evident after pulsing at mean pressure of 50 and 100 mm Hg, but not at 150 and 200 mm Hg, and was caused by the pulsatile change in diameter or deformation and not by the pulsatile change in wall tension. The magnitude of the effect was directly related to the duration of the pulsing period and to the frequency and amplitude of the pressure pulses. The sensitization could not be explained by increased diameter (sonomicrometers) or strain of the carotid sinus at the same pressure after pulsing; thus, there was an increase in "strain sensitivity" that outlasted the period of pulsing by up to several minutes. In most experiments the shift from static to pulsatile pressure at 50 and 100 mm Hg caused an increase in nerve activity, yet sensitization occurred after pulsing when one would have expected postexcitatory hyperpolarization or depression of activity upon return to static pressure. The sensitization was not caused by the release of prostacyclin from the endothelium since it was not reduced after endothelial denudation or inhibition of cyclooxygenase with indomethacin (30-80 microM) or ibuprofen (250 microM). We speculate that sensitization of baroreceptors by pulsatile pressure may contribute to the decreased sympathetic activity after periods of elevated pulse pressure (e.g., after exercise). We also propose that the decreased sensitivity of baroreceptors after acute elevation of arterial pressure (acute resetting) may be offset in part by the sensitizing effect of increased pulsatile stretch.


From drug response profiling to target addiction scoring in cancer cell models.
Deconvoluting the molecular target signals behind observed drug response phenotypes is an important part of phenotype-based drug discovery and repurposing efforts. We demonstrate here how our network-based deconvolution approach, named target addiction score (TAS), provides insights into the functional importance of druggable protein targets in cell-based drug sensitivity testing experiments. Using cancer cell line profiling data sets, we constructed a functional classification across 107 cancer cell models, based on their common and unique target addiction signatures. The pan-cancer addiction correlations could not be explained by the tissue of origin, and only correlated in part with molecular and genomic signatures of the heterogeneous cancer cells. The TAS-based cancer cell classification was also shown to be robust to drug response data resampling, as well as predictive of the transcriptomic patterns in an independent set of cancer cells that shared similar addiction signatures with the 107 cancers. The critical protein targets identified by the integrated approach were also shown to have clinically relevant mutation frequencies in patients with various cancer subtypes, including not only well-established pan-cancer genes, such as PTEN tumor suppressor, but also a number of targets that are less frequently mutated in specific cancer types, including ABL1 oncoprotein in acute myeloid leukemia. An application to leukemia patient primary cell models demonstrated how the target deconvolution approach offers functional insights into patient-specific addiction patterns, such as those indicative of their receptor-type tyrosine-protein kinase FLT3 internal tandem duplication (FLT3-ITD) status and co-addiction partners, which may lead to clinically actionable, personalized drug treatment developments. To promote its application to the future drug testing studies, we have made available an open-source implementation of the TAS calculation in the form of a stand-alone R package.


Doctors and nurses once more--an alternative to May.
It is argued that promissory obligation arising from the contract of employment offers a simpler and less contentious explanation and justification of the doctor-nurse relationship at work, than does May's proposal of second-order reasons. The second-order reason position is rejected as the norm for that relationship, and in the exceptional case, where it is admitted, shared employee status is identified as primary validator of a doctor as locus of rational authority. Finally, a brief case is made for a more precise vocabulary to describe the doctor-nurse relationship, as a contribution to more collegial relations.


Masitinib decreases signs of canine atopic dermatitis: a multicentre, randomized, double-blind, placebo-controlled phase 3 trial.
This study investigated the efficacy and safety of masitinib, a selective tyrosine kinase inhibitor capable of downregulating mast cell functions, for treatment of canine atopic dermatitis (CAD). Dogs with confirmed CAD received masitinib at 12.5 mg/kg/day (n = 202) or control (n = 104) for 12 weeks. A reduction in CAD Extent and Severity Index (CADESI-02) score of ≥ 50% at week 12 was observed in 61% of masitinib-treated dogs versus 35% of control dogs (P < 0.001), according to the modified intent-to-treat population. For dogs resistant to ciclosporin and/or corticosteroids (60% of the study population), CADESI-02 response rates were 60 versus 31%, respectively (P = 0.004). The mean reduction in pruritus score of severely pruritic dogs was 46 versus 29%, respectively (P = 0.045). Furthermore, 65% of owners with severely pruritic dogs assessed masitinib efficacy as good/excellent versus 35% control (P = 0.05). Overall, 63% of investigators assessed masitinib efficacy as good/excellent versus 35% control (P < 0.001). Premature discontinuations from the modified intent-to-treat population (28.2% masitinib versus 26.0% control) were mainly due to adverse events (13.4 versus 4.8%, respectively) or lack of efficacy (12.4 versus 18.3%, respectively). In total, 13.2% dogs presented with severe adverse events (16.0% masitinib versus 7.7% control). Masitinib showed a risk of reversible protein loss, although regular surveillance of blood albumin and proteinuria allowed for discontinuation of treatment while the dog was still clinically asymptomatic. Masitinib proved to be an effective and mostly well-tolerated treatment of CAD, including severe and refractory cases, with medically manageable adverse effects.


Seizure-induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling.
Increased expression of drug efflux transporters at the blood-brain barrier accompanies epileptic seizures and complicates therapy with antiepileptic drugs. This study is concerned with identifying mechanistic links that connect seizure activity to increased P-glycoprotein expression at the blood-brain barrier. In this regard, we tested the hypothesis that seizures increase brain extracellular glutamate, which signals through an N-methyl-d-aspartate (NMDA) receptor and cyclooxygenase-2 (COX-2) in brain capillaries to increase blood-brain barrier P-glycoprotein expression. Consistent with this hypothesis, exposing isolated rat or mouse brain capillaries to glutamate for 15 to 30 min increased P-glycoprotein expression and transport activity hours later. These increases were blocked by 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate) (MK-801), an NMDA receptor antagonist, and by celecoxib, a selective COX-2 inhibitor; no such glutamate-induced increases were seen in brain capillaries from COX-2-null mice. In rats, intracerebral microinjection of glutamate caused locally increased P-glycoprotein expression in brain capillaries. Moreover, using a pilocarpine status epilepticus rat model, we observed seizure-induced increases in capillary P-glycoprotein expression that were attenuated by administration of indomethacin, a COX inhibitor. Our findings suggest that brain uptake of some antiepileptic drugs can be enhanced through COX-2 inhibition. Moreover, they provide insight into one mechanism that underlies drug resistance in epilepsy and possibly other central nervous system disorders.


Effect of different operating modes and biomass concentrations on the recovery of recombinant hepatitis B core antigen from thermal-treated unclarified Escherichia coli feedstock.
Expanded bed adsorption chromatography (EBAC) is a single pass operation that has been used as primary capture step in various protein purifications. The most common problem in EBAC is often associated with successful formation of a stable fluidized bed during the absorption stage, which is critically dependent on parameters such as liquid velocity, bed height, particle (adsorbent) size and density as well as design of column and type of flow distributor. In this study, residence time distribution (RTD) test using acetone as non-binding tracer acetone was performed to evaluate liquid dispersion characteristics of the EBAC system. A high B(o) number was obtained indicating the liquid dispersion in the system employed is very minimal and the liquid flow within the bed was close to plug flow, which mimics a packed bed chromatography system. Evaluation on the effect of flow velocities and bed height on the performance of Streamline DEAE using feedstock containing heat-treated crude Escherichia coli homogenate of different biomass concentrations was carried out in this study. The advantages and disadvantages as well as the problems encountered during recovery of HBcAg with aforementioned parameters are also discussed in this paper.


Arabidopsis thaliana AMY3 is a unique redox-regulated chloroplastic α-amylase.
α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp(666)), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5-8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys(499) and Cys(587) is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.


Effects of daily irrigation on corneal epithelial permeability and adverse events with silicone hydrogel contact lens continuous wear.
To determine whether daily irrigation with sterile saline solution during silicone hydrogel (SiH) contact lens continuous wear (CW) can mitigate increases in corneal epithelial permeability (Pdc) and reduce the risk of adverse events. In this study, 161 non-contact lens wearers were fit with SiH contact lenses and randomized to either a treatment (n = 81) or control (n = 80) group for 30-day CW. Subjects in the treatment group irrigated every morning and whenever dryness symptoms occurred; subjects in the control group did not. Mixed effects models were employed to assess the changes in Pdc, and survival analysis methods were employed to estimate the risk for adverse events. Epithelial permeability increased over 30-day CW with SiH lenses (P = 0.001). Risk of inflammatory (odds ratio [OR] = 1.94) and lens-induced (OR = 1.90) adverse events increased with irrigation; these events also occurred sooner, on average, in the irrigation group. Although the overall risk for Asians was higher than for non-Asians, irrigation had no significant impact on risk among Asians, but significantly increased risk among non-Asians. Thirty-day CW results in a measurable increase in epithelial permeability. Risk of inflammatory and lens-induced adverse events also increase over time during CW. Daily irrigation with sterile saline solution did not mitigate increases in epithelial permeability or decrease risk of adverse events.


Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2'-O-transphosphorylation.
Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2'-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the (18)O KIEs on the 2'O nucleophile, 5'O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium- or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5'O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/Lys41), together with proton transfer from His119, render departure of the 5'O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2'-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure.


[Satisfaction with hospital care among diabetic outpatients and its associated factors. Secondary use of official statistics].
Generalizable data on current satisfaction levels are required to establish a scientific basis for the political advancement of measures to improve satisfaction with hospital care among patients with diabetes. The present study made secondary use of existing official statistics in order to demonstrate the range of satisfaction levels with hospital care among diabetic outpatients and to closely examine related factors. Data sets that consolidated the Patient Survey, the Survey of Medical Care Institutions, and the Patient Behavior Survey (all from 2008) were created. Shared medical institution survey reference numbers were used to consolidate the data from the Patient Survey and the Survey of Medical Care Institutions, and in addition, sex and date of birth were used to consolidate the Patient Behavior Survey data. The range of satisfaction levels with hospital care among diabetic outpatients was investigated along with any relationship with the following potentially related factors: visitation status (first or repeat examination); waiting time until examination; examination duration; care-seeking status (any use of other medical facilities, etc.); diabetic complications; other complications; coverage under the Public Assistance Act; smoking cessation outpatient services; hospitals that specialized in treating diabetes (metabolic medicine); medical care on Saturday, Sunday, and public holidays; and provision of health checkups. Overall, 62.3% of diabetic outpatients were either fairly or extremely satisfied with their hospital care, whereas 5.6% expressed dissatisfaction. Satisfaction levels with hospital care were found to be significantly related to visitation status, waiting time until examination, examination duration, care-seeking status, and Saturday medical care. Multivariate analysis with the factors demonstrated to be significantly related to satisfaction revealed significant relationships between high satisfaction levels and repeat examinations, short waiting times, no use of any other medical facilities, and long examinations. Consolidating official statistics from multiple sources indicated the range of satisfaction levels with hospital care among diabetic outpatients and facilitated the clarification of factors affecting satisfaction. Reducing waiting times and ensuring sufficient time spent on examinations are important for increasing satisfaction levels with hospital care among patients with diabetes. It is hoped that official statistics can be further applied to many future public health policy studies.


The need for closure in caregivers of people with psychosis.
The aim of the study was to determine how carer need for closure relates to expressed emotion. It also examined the links between carer need for closure and patient functioning including patient need for closure. In a cross-sectional study, 70 caregivers of patients with psychosis completed the Need for Closure Scale (NFCS), the Camberwell Family Interview (CFI) and measures of distress, burden, coping and social network. The NFCS was assessed in terms of its two primary dimensions: a need for simple structure (NFSS) and Decisiveness. Patients also completed measures of psychotic symptoms and affect, and in 50 matched caregiver patient dyads, direct comparisons were undertaken between caregiver and patient NFCS scores. No links were found between caregiver NFC and EE in this predominately low EE sample. More decisive carers had higher levels of self esteem, were less distressed, and resorted less to avoidant coping. The need for simple structure was greater in carers who lacked a confidante. As predicted, patients reported significantly higher NFSS and lower Decisiveness scores than carers, but no relationship was observed between caregiver NFC and patient symptoms of psychosis. Carers reporting confident decision making were also more likely to report adaptive functioning in terms of having lower levels of avoidant coping and distress, and higher levels of self esteem. The results suggest that this style of thinking might be a helpful way of coping with some of the difficulties involved in caring for someone with psychosis. D


Impaired axonal regeneration in alpha7 integrin-deficient mice.
The interplay between growing axons and the extracellular substrate is pivotal for directing axonal outgrowth during development and regeneration. Here we show an important role for the neuronal cell adhesion molecule alpha7beta1 integrin during peripheral nerve regeneration. Axotomy led to a strong increase of this integrin on regenerating motor and sensory neurons, but not on the normally nonregenerating CNS neurons. alpha7 and beta1 subunits were present on the axons and their growth cones in the regenerating facial nerve. Transgenic deletion of the alpha7 subunit caused a significant reduction of axonal elongation. The associated delay in the reinnervation of the whiskerpad, a peripheral target of the facial motor neurons, points to an important role for this integrin in the successful execution of axonal regeneration.


Current use of beta blockers in patients with reactive airway disease who are hospitalized with acute coronary syndromes.
Beta blockers have a proven benefit in the management of patients with acute coronary syndromes (ACS) and for secondary prevention of coronary events. Current guidelines list such reactive airway diseases (RADs) such as asthma and chronic obstructive pulmonary disease as relative contraindications to beta-blocker use. However, the co-morbid burden of RAD and coronary heart disease is substantial, and data suggest that the treatment benefit of beta blockers is shared by patients with RAD. The Get with the Guidelines (GWTG) database was used to evaluate use of beta blockers within 24 hours of admission and at discharge in patients with ACS with (n = 12,967) and without (n = 81,140) a history of RAD. Data were collected in 435 hospitals between January 2000 and September 2006. A multivariable logistic regression model was used to determine predictors of beta-blocker treatment. In patients with no RAD history, beta-blocker prescription rates were 78.3% at admission and 88.7% at discharge; in patients with a RAD history, rates were 65.6% at admission and 77.2% at discharge. Compared with patients with no history of RAD, patients with a history of RAD were 42% less likely (odds ratio 0.58, confidence interval 0.54 to 0.62, p <0.0001) to receive a beta blocker upon admission and 55% less likely (odds ratio 0.45, confidence interval 0.41 to 0.48, p <0.0001) to receive a beta blocker at discharge in multivariable analysis. Among all other clinical factors, RAD history was the most significant predictor of likelihood of not receiving a beta blocker at admission or discharge. Receipt of beta blockers within 24 hours after admission was associated with a lower in-hospital mortality rate for patients with RAD (odds ratio = 0.52, p <0.001) and for patients without RAD (odds ratio = 0.38, p <0.001). Careful assessment of beta-blocker safety and RAD severity by physicians is needed to improve beta-blocker prescription rates in this large group of patients with ACS.


Correlated Electrochemical and Optical Detection Reveals the Chemical Reactivity of Individual Silver Nanoparticles.
Electrochemical (EC) impacts of single nanoparticles (NPs) on an ultramicroelectrode are coupled with optics to identify chemical processes at the level of individual NPs. While the EC signals characterize the charge transfer process, the optical monitoring gives a complementary picture of the transport and chemical transformation of the NPs. This is illustrated in the case of electrodissolution of Ag NPs. In the simplest case, the optically monitored dissolution of individual NPs is synchronized with individual EC spikes. Optics then validates in situ the concept of EC nanoimpacts for sizing and counting of NPs. Chemical complexity is introduced by using a precipitating agent, SCN(-), which tunes the overall electrodissolution kinetics. Particularly, the charge transfer and dissolution steps occur sequentially as the synchronicity between the EC and optical signals is lost. This demonstrates the level of complexity that can be revealed from such electrochemistry/optics coupling.


A novel essential fission yeast gene pad1+ positively regulates pap1(+)-dependent transcription and is implicated in the maintenance of chromosome structure.
Fission yeast pap1+ gene encodes an AP-1-like transcription factor, whose overexpression can confer resistance to staurosporine, a protein kinase inhibitor. We have previously identified a target gene (p25) for pap1+, and shown that, crm1+, which is required for maintenance of higher order chromosome structure, negatively regulates pap1-dependent transcription. In this study, we have characterized a novel gene, pad1+, which was isolated as a multicopy plasmid capable of conferring staurosporine-resistance. We showed that high copy pad1+ induces transcriptional activation of the p25 gene and that the induction by pad1+ is dependent on the pap1+ gene. Furthermore, a cis-element analysis of the 5'-region of the p25 gene showed that two elements (an AP-1 site and a 14 bp palindrome sequence) where pap1 binds in vitro is essential for the induction by pad1+. These results indicate that pad1 can positively regulate pap1-dependent transcription. Through an electromobility shift assay we showed that overexpression of pad1+ is not capable of enhancing the DNA-binding activity of pap1 directly. The pad1+ gene encodes a 35 kDa protein that has significant identity (68%) to Caenorhabditis elegans F37A4.5, and is also similar to mouse Mov34 and human C6.1A. Gene disruption experiments have demonstrated that pad1+ is essential for viability. A disruption mutant of pad1+ obtained after spore germination exhibited an elongated cell body with abberantly folded chromosomes. A mitotic plasmid loss experiment also produced similar cells having an abnormal chromosome structure. These suggest that pad1+ may play an important role in higher order chromosome structure. Taken concurrently with our previous results, two essential genes pad1+ and crm1+ regulate pap1-dependent transcription; pad1+ and crm1+ are positive and negative regulators, respectively.


[Overview and perspectives of mesenchymal stem cell therapy in intensive care medicine].
Development of innovative therapies in intensive care medicine is particularly important since diseases as sepsis, acute respiratory distress syndrome (ARDS) and acute renal injury (AKI) have an elevated morbidity and mortality in spite of current gold-standard approaches. Mesenchymal stem cells (MSC) may have a promising role due to their properties in immunomodulation, tissue reparation and microbial clearance. Preclinical data and results of a systematic review of PubMed, PMC and ClinicalTrials.gov have been included to review the role of MSC therapy in sepsis, ARDS and AKI. A description of MSC biology, sources and benefits in preclinical models was included. A phase I/II clinical trial (RCT) is recruiting neutropenic patients with septic shock. In ARDS, the START trial (Stem cells in ARDS Treatment) is a phase I/II study of bone marrow-derived human MSC (hMSC) that is currently recruiting patients. In AKI, a phase I study has demonstrated the safety of hMSCs infusion in patients undergoing cardiac surgery with high risk to develop AKI. A phase II study is still active. The results of these studies will determine the real feasibility of MSC therapy in critically ill patients.


Timing of allogeneic stem cell transplantation for myelodysplastic syndromes and aplastic anemia.
Allogeneic hematopoietic stem cell transplantation (HSCT) for myelodysplastic syndrome (MDS) is a potentially curative procedure, but is associated with a significant risk of morbidity and mortality. With the recent approval of disease-modifying agents, the appropriate timing of allogeneic HSCT needs to be addressed. Similarly, the optimal use of these disease-modifying agents before HSCT needs to be determined. In severe aplastic anemia, HSCT is a proven cure, but HLA-matched sibling donors are found in fewer than 25% of newly diagnosed patients. The use of early unrelated donor HSCT is an evolving concept that will become more accepted as improvements in HSCT outcomes continue.


A truce with neutral theory: local deterministic factors, species traits and dispersal limitation together determine patterns of diversity in stream invertebrates.
1. Studies seeking to explain local patterns of diversity have typically relied on niche explanations, reflected in correlations with local environmental conditions, or neutral theory, invoking dispersal processes and speciation. 2. We used macroinvertebrate community data from 10 streams that varied independently in local ecological conditions and spatial proximity. Neutral theory predicts that similarity in communities will be negatively associated with distance between sites, while niche theory suggests that community similarity will be positively associated with similarity in local ecological conditions. 3. Similarity in total invertebrate, grazer and predator assemblages showed negative relationships with distance and, for grazers and predators, positive relationships with local ecological conditions. However, the best model predicting community similarity in all three cases included aspects of both local ecological conditions and distance between sites. 4. When assemblages were analysed according to dispersal ability, high-dispersal species were shown to be freely accessing all sites and community similarity was not well predicted by either local ecology or spatial separation. Assemblages of species with low and moderate dispersal ability were best predicted by combined models, including distance between sites and local ecological factors. 5. The results suggest that the perceived dichotomy between neutral and local environmental processes in determining local patterns of diversity may not be useful. Neutral and niche processes structured these communities differentially depending on trophic level and species traits. 6. We emphasize the potential for both dispersal processes and local environmental conditions to explain local patterns of diversity.


Abnormal expression of collagen IV in lens activates unfolded protein response resulting in cataract.
Human diseases caused by mutations in extracellular matrix genes are often associated with an increased risk of cataract and lens capsular rupture. However, the underlying mechanisms of cataract pathogenesis in these conditions are still unknown. Using two different mouse models, we show that the accumulation of collagen chains in the secretory pathway activates the stress signaling pathway termed unfolded protein response (UPR). Transgenic mice expressing ectopic Col4a3 and Col4a4 genes in the lens exhibited activation of IRE1, ATF6, and PERK associated with expansion of the endoplasmic reticulum and attenuation of general protein translation. The expression of the transgenes had adverse effects on lens fiber cell differentiation and eventually induced cell death in a group of transgenic fiber cells. In Col4a1(+/Deltaex40) mutant mice, the accumulation of mutant chains also caused low levels of UPR activation. However, cell death was not induced in mutant lenses, suggesting that low levels of UPR activation are not proapoptotic. Collectively, the results provide in vivo evidence for a role of UPR in cataract formation in response to accumulation of terminally unfolded proteins in the endoplasmic reticulum.


Algorithm aversion: people erroneously avoid algorithms after seeing them err.
Research shows that evidence-based algorithms more accurately predict the future than do human forecasters. Yet when forecasters are deciding whether to use a human forecaster or a statistical algorithm, they often choose the human forecaster. This phenomenon, which we call algorithm aversion, is costly, and it is important to understand its causes. We show that people are especially averse to algorithmic forecasters after seeing them perform, even when they see them outperform a human forecaster. This is because people more quickly lose confidence in algorithmic than human forecasters after seeing them make the same mistake. In 5 studies, participants either saw an algorithm make forecasts, a human make forecasts, both, or neither. They then decided whether to tie their incentives to the future predictions of the algorithm or the human. Participants who saw the algorithm perform were less confident in it, and less likely to choose it over an inferior human forecaster. This was true even among those who saw the algorithm outperform the human.


Identification of dietary patterns and their relationships with general and oral health in the very old.
Little is known about the dietary patterns of Asian populations aged >=85 years and their associated factors. Thus, we aimed to (1) identify these dietary patterns and (2) clarify the relationships between the dietary pattern and health outcomes in a community-dwelling very old population. The Tokyo Oldest Old Survey on Total Health study is an observational cohort study comprising 512 Japanese subjects (women, n=288; men, n=224; age, 87.8±2.2 years). Dietary patterns were assessed by principal component analysis using a brief self-administered diet history questionnaire. Barthel index, Mini-Mental State Examination, and oral health status [maximum occlusal force (MOF), denture use, and dentulous / edentulous state] were also measured. Two dietary patterns were identified. The first factor component "traditional Japanese" was characterized by a high consumption of vegetables, seaweed, legumes, and fish. The second factor component "noodles and confectioneries" was characterized by a high consumption of noodles, confectioneries, and non-alcoholic beverages. Multivariable analysis showed that the "traditional Japanese" dietary pattern was inversely associated with dentulous state (OR: 0.53; 95% CI: 0.34-0.82), the lowest tertile of MOF (OR: 0.64; 95% CI: 0.42-0.99), and denture use (OR: 2.42; 95% CI: 1.26- 4.63) even after adjustment for potential confounders. Furthermore, the "noodles and confectioneries" dietary pattern was inversely associated with the lowest tertile of MOF (OR: 0.62; 95% CI: 0.40-0.94). However, there were no significant associations between these dietary patterns and disability or cognitive function. We identified two dietary patterns in the very old population, which were associated with oral health status.


Bronchiolitis obliterans syndrome: the final frontier for lung transplantation.
Bronchiolitis obliterans syndrome (BOS) is a form of chronic lung allograft dysfunction that affects a majority of lung transplant recipients and is the principal factor limiting long-term transplant survival. BOS is characterized by progressive airflow obstruction unexplained by acute rejection, infection, or other coexistent condition. Although BOS is a proven useful clinical syndrome that identifies patients at increased risk for death, its clinical course and underlying causative factors are now recognized to be increasingly heterogeneous. Regardless of the clinical history, the primary pathologic correlate of BOS is bronchiolitis obliterans, a condition of intraluminal airway fibrosis. This article highlights the body of developing research illustrating the mechanisms by which BOS is mediated, including alloimmune reactivity, the emerging roles of humoral and autoimmunity, activation of innate immune cells, and response to nonimmune-related allograft insults, such as infection and aspiration. In addition, we underscore emerging clinical implications and promising future translational research directions that have the potential to advance our knowledge and improve patient outcomes.


Hyperoxia decreases matrix metalloproteinase-9 and increases tissue inhibitor of matrix metalloproteinase-1 protein in the newborn rat lung: association with arrested alveolarization.
Matrix metalloproteinases (MMP) are likely effectors of normal lung development, especially branching morphogenesis, angiogenesis, and extracellular matrix degradation. Because hyperoxia exposure (>95% O(2)) from d 4 to 14 in newborn rat pups leads to arrest of alveolarization and mimics newborn chronic lung disease, we tested whether hyperoxia altered MMP-2 and -9 mRNA, protein, and enzymatic activity, and the mRNA and protein expression of the endogenous tissue inhibitor of MMP, TIMP-1. No changes due to hyperoxia exposure were observed in MMP-2 mRNA or pro-enzyme (72 kD) protein levels between d 6 and 14, although the overall protein mass and zymographic activity of the active (68 kD) enzyme were diminished (p < 0.05, ANOVA). However, hyperoxia significantly decreased levels of MMP-9 mRNA and pro-MMP-9 protein and diminished overall MMP-9 pro-enzyme activity. TIMP-1 mRNA was not elevated by hyperoxia until d 14, but protein levels were significantly (p < 0.001) elevated by hyperoxia from d 9 to 14. To estimate the potential of MMP inhibition to arrest alveolarization, administration of doxycycline (20 mg/kg, twice daily by gavage), a pan-MMP proteolysis inhibitor, arrested lung alveolarization. We conclude that hyperoxia decreases MMP-9 mRNA, protein, and activity and elevates TIMP-1 protein, and these changes have the potential to contribute to the arrest of normal lung development.


Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications.
The widespread use of genome-wide diagnostic screening methods has greatly increased the frequency with which incidental (but possibly pathogenic) copy number changes affecting single genes are detected. These findings require validation to allow appropriate clinical management. Deletion variants can usually be readily validated using a range of short-read next-generation sequencing (NGS) strategies, but the characterization of duplication variants at nucleotide resolution remains challenging. This presents diagnostic problems, since pathogenicity cannot generally be assessed without knowing the structure of the variant. We have used a novel Cas9 enrichment strategy, in combination with long-read single-molecule nanopore sequencing, to address this need. We describe the nucleotide-level resolution of two problematic cases, both of whom presented with neurodevelopmental problems and were initially investigated by array CGH. In the first case, an incidental 1.7-kb imbalance involving a partial duplication of VHL exon 3 was detected. This variant was inherited from the patient's father, who had a history of renal cancer at 38 years. In the second case, an incidental ~200-kb de novo duplication that included DMD exons 30-44 was resolved. In both cases, the long-read data yielded sufficient information to enable Sanger sequencing to define the rearrangement breakpoints, and creation of breakpoint-spanning PCR assays suitable for testing of relatives. Our Cas9 enrichment and nanopore sequencing approach can be readily adopted by molecular diagnostic laboratories for cost-effective and rapid characterization of challenging duplication-containing alleles. We also anticipate that in future this method may prove useful for characterizing acquired translocations in tumor cells, and for precisely identifying transgene integration sites in mouse models.


Distribution of viral RNA in the spinal cord of DBA/2 mice developing biphasic paralysis following infection with the D variant of encephalomyocarditis virus (EMC-D).
DBA/2 mice infected with the D variant of encephalomyocarditis virus (EMC-D) (10(1) PFU/head) developed biphasic hind limb paralysis. As a first step in clarifying its pathogenesis, we examined the distribution of viral RNA in the spinal cord using in situ hybridization. At 3 days post inoculation (DPI), in the spinal cord of mice showing slight paralysis, viral RNA was observed in capillary endothelial cells and a few adjacent glia cells in the funiculus lateralis from thoracic to lumbar enlargement. At 7 DPI, in the spinal cord of mice showing apparent paralysis, viral RNA was observed in a larger number of glia cells in the demyelinated lesion associated with infiltration of macrophages in the funiculus lateralis and in a small number of degenerated neurons in the cornu ventrale. In the funiculus lateralis, viral RNA could not be observed after 28 DPI. On the other hand, viral RNA was observed in degenerated neurons in the cornu ventrale of mice showing the second phase paralysis at 42 DPI. Many CD4+T cells infiltrated around these degenerated neurons. These results suggest that: (1) the viral entry zone was the capillary endothelial cells in the funiculus lateralis; (2) first phase paralysis was due to demyelination caused by EMC-D and associated with macrophage infiltration; (3) second phase paralysis was due to degeneration of motor neurons bearing viral RNA associated with infiltration by CD4+T cells.


Determinants of compliance with drinking water standards in rural Puerto Rico between 1996 and 2000: a multilevel approach.
Two hundred and thirty nine (239) drinking water systems in Puerto Rico are not connected to the Puerto Rico Aqueducts and Sewers Authority (PRASA), and are thus known as Non-PRASA drinking water systems. Population served estimates by Non-PRASA systems are in the 100,000 to 300,000 range. To identify the determinants of compliance with drinking water standards by rural drinking water systems in Puerto Rico. To identify the best analytical methods for studying the problem of non-compliance with drinking water standards in Puerto Rico and its generalization to similar communities elsewhere. We reviewed capacity development and drinking water system evaluations performed by governmental and academic institutions between 1993 and 2004. Community and system variables were used to fit a multilevel model to predict compliance with drinking water standards. Data was obtained from the Environmental Protection Agency' Safe Drinking Water Information System and the Puerto Rico Health Department drinking water database for 231 systems, serving 90,000 persons. There was an 11% increase in compliance (1996 = 4%; 2000=15%), a decrease of 13,634 people served by non-compliant systems (1996 = 86,169; 2000 = 72,535) and a 6% decrease in the number of non-compliant systems which had installed treatment equipment (1996 = 93%; 2000=87%). The prevalence of compliance among those systems that had installed treatment equipment was higher than among those systems that did not have treatment equipment, after adjusting by the time period (est. POR = 2.2, 95% CI, 1.40 - 3.44). Our findings suggest alternative public health strategies are needed to ensure sustained safe water capacity in rural communities.


Bioethics: why philosophy is essential for progress.
It is the JME's 40th anniversary and my 20th anniversary working in the field. I reflect on the nature of bioethics and medical ethics. I argue that both bioethics and medical ethics together have, in many ways, failed as fields. My diagnosis is that better philosophy is needed. I give some examples of the importance of philosophy to bioethics. I focus mostly on the failure of ethics in research and organ transplantation, although I also consider genetic selection, enhancement, cloning, futility, disability and other topics. I do not consider any topic comprehensively or systematically or address the many reasonable objections to my arguments. Rather, I seek to illustrate why philosophical analysis and argument remain as important as ever to progress in bioethics and medical ethics.