The CAP study, evaluation of integrated universal and selective prevention strategies for youth alcohol misuse: study protocol of a cluster randomized controlled trial.
Alcohol misuse amongst young people is a serious concern. The need for effective prevention is clear, yet there appear to be few evidenced-based programs that prevent alcohol misuse and none that target both high and low-risk youth. The CAP study addresses this gap by evaluating the efficacy of an integrated approach to alcohol misuse prevention, which combines the effective universal internet-based Climate Schools program with the effective selective personality-targeted Preventure program. This article describes the development and protocol of the CAP study which aims to prevent alcohol misuse and related harms in Australian adolescents. A cluster randomized controlled trial (RCT) is being conducted with Year 8 students aged 13 to 14-years-old from 27 secondary schools in New South Wales and Victoria, Australia. Blocked randomisation was used to assign schools to one of four groups; Climate Schools only, Preventure only, CAP (Climate Schools and Preventure), or Control (alcohol, drug and health education as usual). The primary outcomes of the trial will be the uptake and harmful use of alcohol and alcohol related harms. Secondary outcomes will include alcohol and cannabis related knowledge, cannabis related harms, intentions to use, and mental health symptomatology. All participants will complete assessments on five occasions; baseline; immediately post intervention, and at 12, 24 and 36 months post baseline. This study protocol presents the design and current implementation of a cluster RCT to evaluate the efficacy of the CAP study; an integrated universal and selective approach to prevent alcohol use and related harms among adolescents. Compared to students who receive the stand-alone universal Climate Schools program or alcohol and drug education as usual (Controls), we expect the students who receive the CAP intervention to have significantly less uptake of alcohol use, a reduction in average alcohol consumption, a reduction in frequency of binge drinking, and a reduction in alcohol related harms. This trial is registered with the Australian and New Zealand Clinical Trials registry, ACTRN12612000026820.


Active maturation-promoting factor is present in mature mouse oocytes.
Cytoplasmic extracts of meiotically mature mouse oocytes were injected into immature Xenopus laevis oocytes, which underwent germinal vesicle breakdown within 2 h. Germinal vesicle breakdown was not inhibited by incubation of the Xenopus oocytes in cycloheximide (20 micrograms/ml). Identically prepared extracts of meiotically immature mouse oocytes, arrested at the germinal vesicle stage by dibutyryl cyclic AMP (100 micrograms/ml), did not induce germinal vesicle breakdown in Xenopus oocytes. The results show that maturation-promoting factor activity appears during the course of oocyte maturation in the mouse.


A study on L2-loss (squared hinge-loss) multiclass SVM.
Crammer and Singer's method is one of the most popular multiclass support vector machines (SVMs). It considers L1 loss (hinge loss) in a complicated optimization problem. In SVM, squared hinge loss (L2 loss) is a common alternative to L1 loss, but surprisingly we have not seen any paper studying the details of Crammer and Singer's method using L2 loss. In this letter, we conduct a thorough investigation. We show that the derivation is not trivial and has some subtle differences from the L1 case. Details provided in this work can be a useful reference for those who intend to use Crammer and Singer's method with L2 loss. They do not need a tedious process to derive everything by themselves. Furthermore, we present some new results on and discussion of both L1- and L2-loss formulations.


Patination of Cultural Flints: Flint artifacts can be dated by cortical changes in mineralogy and texture.
All flints containing unstable impurities are susceptible to patination. The rate of patination varies with many factors: (i) the texture and microstructure of the flint; (ii) its permeability; (iii) the kind, proportion and distribution of impurities; and (iv) environmental factors, such as temperature and soil chemistry. The thickness of the patina varies also with time. Two contrasting types of patina can develop: a chalky white patina and a ferruginous brown patina. Both types are observable primarily as a color change, and study of these types is facilitated by a clear understanding of the causes of color in flint. The color of most flints is the result of repeated refraction and reflection of light at numerous intergranular surfaces, whereby part of the light is internally absorbed and part is reflected back to the observer. The ratio of reflected to absorbed light governs the lightness of the color, or its value. The preferential absorption of certain wavelengths by natural pigments (such as iron oxide and hydrous iron oxide) disseminated through the flint determine the hue of the color. The color changes produced during patination relate to changes in texture and impurity content occasioned by the attack of weathering agents on impurities in the flint. The creation of voids by the dissolution and leaching of carbonates, the loosening of quartz crystallites, and the dispersal of clays all modify the reflectivity of the flint. Chemical changes involving the epigments, their dispersal along intergranular surfaces, or removal by leachig modify both reflectivity and capacity to preferentially absorb. Attempts to correlate patina thickness with age, and thus to use flint patinae chronometrically, have proven unsatisfactory because other factors, whose importance in some cases exceeds that of age, have not been taken into account. The texture and microstructure of flint, its permeability, and the kind, proportion, and distribution of impurities can be evaluated by regular petrographic techniques. Environmental factors can be assumed constant for artifacts from the same types of soil in a given climatic region. Only after allowances have been made for these additional variables does the age-dependence of flint patination become clear.


Measurement of the local food environment: a comparison of existing data sources.
Studying the relation between the residential environment and health requires valid, reliable, and cost-effective methods to collect data on residential environments. This 2002 study compared the level of agreement between measures of the presence of neighborhood businesses drawn from 2 common sources of data used for research on the built environment and health: listings of businesses from commercial databases and direct observations of city blocks by raters. Kappa statistics were calculated for 6 types of businesses-drugstores, liquor stores, bars, convenience stores, restaurants, and grocers-located on 1,663 city blocks in Chicago, Illinois. Logistic regressions estimated whether disagreement between measurement methods was systematically correlated with the socioeconomic and demographic characteristics of neighborhoods. Levels of agreement between the 2 sources were relatively high, with significant (P < 0.001) kappa statistics for each business type ranging from 0.32 to 0.70. Most business types were more likely to be reported by direct observations than in the commercial database listings. Disagreement between the 2 sources was not significantly correlated with the socioeconomic and demographic characteristics of neighborhoods. Results suggest that researchers should have reasonable confidence using whichever method (or combination of methods) is most cost-effective and theoretically appropriate for their research design.


Personal, professional and practice development: clinical supervision.
This article investigates current research concerns in relation to clinical supervision and offers a rationale for using case studies as a means of evaluation. Three case studies are used to illustrate how development and growth can occur as a result of clinical supervision. The use of case studies as a means of evaluation is analysed. Case studies provide unique insights into the dynamics and processes of clinical supervision. Any improvement in practice ought to bring about improvements in client care, thus case studies can add to the accumulating, qualitative data that support the link between clinical supervision and improved client care. This article recommends that more case studies should be carried out and that the findings are compiled on a centralized database in order that evidence supporting the widespread use of clinical supervision is readily accessible.


Widespread Occurrence of the Perfect Stage of Powdery Mildew Caused by Erysiphe polygoni on Sugar Beets in Nebraska.
Powdery mildew, caused by Erysiphe polygoni DC (synonym E. betae [Vanha] Weltzien), has been a sporadic and relatively minor problem for sugar beet (Beta vulgaris L.) growers in western Nebraska. Yield losses in this region have been limited, in part because of the use of effective fungicides, but also because infection occurs late enough in the season that treatment has often been unnecessary. The perfect stage had been reported only once in the United States until 2001-2002 when it was identified from Idaho and Colorado (1). The teleomorph was also noted from several fields in Scotts Bluff County in Nebraska in October 2002. The first appearance of the disease in 2003 occurred during the second week of August within five miles of the fields where the perfect stage was noted in 2002. On the basis of these observations, a survey was conducted between mid-August and mid-October to map the appearance and distribution of the perfect stage of E. polygoni within the Nebraska Panhandle growing region. During this time, between 45 and 50 fields were surveyed in six Nebraska counties. This represented the majority (70%) of the sugar beet acreage in Nebraska. The first finding of the perfect stage occurred in early September from multiple fields in the vicinity of and including the field where the asexual stage was first reported in August 2003. Ascomata measured 85 to 110 μm with one to four (mostly three) ascospores per ascus, resembling previous pathogen descriptions (2). Subsequently, every other field in the North Platte Valley where the oidial stage had been found also contained the perfect stage by the third week in September, including the Nebraska counties of Scotts Bluff (15 fields) and Morrill (7 fields). Outside the North Platte Valley, powdery mildew was not detected until mid-September and mid-October for the Northern Panhandle (Box Butte County) and Southern Panhandle (Kimball, Banner, and Cheyenne counties) growing areas, respectively. By October 1, the perfect stage was found in 9 of 10 fields exhibiting the disease in the North Panhandle, whereas the perfect stage was not found in the Southern Panhandle before harvest. Over 85% of surveyed fields infected with powdery mildew also harbored the perfect stage (31 of 36). Not only is the new and continued presence of the perfect stage potentially problematic for managing fungicide resistance and developing new cultivars with pathogen resistance (1), but it may also provide a means for overwintering in this area. This could result in earlier and more severe infections that would additionally require uncustomary treatment for powdery mildew control. The unusually early appearance of the disease and the high incidence of the perfect stage in Nebraska fields during 2003 further highlights these concerns and warrants closely monitoring future crops for continued epidemics. References: (1) J. J. Gallian and L. E. Hanson. Plant Dis. 87:200, 2003. (2) E. G. Ruppel. Powdery mildew. Pages 13-15 in: Compendium of Beet Diseases and Insects. E. D. Whitney and J. E. Duffus, eds. The American Phytopathological Society, St. Paul, MN, 1986.


Biomolecular Functionalization of a Nanomaterial To Control Stability and Retention within Live Cells.
Noncovalent hybrids of single-stranded DNA and single-walled carbon nanotubes (SWCNTs) have demonstrated applications in biomedical imaging and sensing due to their enhanced biocompatibility and photostable, environmentally responsive near-infrared (NIR) fluorescence. The fundamental properties of such DNA-SWCNTs have been studied to determine the correlative relationships between oligonucleotide sequence and length, SWCNT species, and the physical attributes of the resultant hybrids. However, intracellular environments introduce harsh conditions that can change the physical identities of the hybrid nanomaterials, thus altering their intrinsic optical properties. Here, through visible and NIR fluorescence imaging in addition to confocal Raman microscopy, we show that the oligonucleotide length controls the relative uptake, intracellular optical stability, and retention of DNA-SWCNTs in mammalian cells. Although the absolute NIR fluorescence intensity of DNA-SWCNTs in murine macrophages increases with increasing oligonucleotide length (from 12 to 60 nucleotides), we found that shorter oligonucleotide DNA-SWCNTs undergo a greater magnitude of spectral shift and are more rapidly internalized and expelled from the cell after 24 h. Furthermore, by labeling the DNA with a fluorophore that dequenches upon removal from the SWCNT surface, we found that shorter oligonucleotide strands are displaced from the SWCNT within the cell, altering the physical identity and changing the fate of the internalized nanomaterial. Finally, through a pharmacological inhibition study, we identified the mechanism of SWCNT expulsion from the cells as lysosomal exocytosis. These findings provide a fundamental understanding of the interactions between SWCNTs and live cells as well as evidence suggesting the ability to control the biological fate of the nanomaterials merely by varying the type of DNA wrapping.


Multiple mechanisms confining RNA polymerase II ubiquitylation to polymerases undergoing transcriptional arrest.
In order to study mechanisms and regulation of RNA polymerase II (RNAPII) ubiquitylation and degradation, highly purified factors were used to reconstitute RNAPII ubiquitylation in vitro. We show that arrested RNAPII elongation complexes are the preferred substrates for ubiquitylation. Accordingly, not only DNA-damage-dependent but also DNA-damage-independent transcriptional arrest results in RNAPII ubiquitylation in vivo. Def1, known to be required for damage-induced degradation of RNAPII, stimulates ubiquitylation of RNAPII only in an elongation complex. Ubiquitylation of RNAPII is dependent on its C-terminal repeat domain (CTD). Moreover, CTD phosphorylation at serine 5, a hallmark of the initiating polymerase, but not at serine 2, a hallmark of the elongating polymerase, completely inhibits ubiquitylation. In agreement with this, ubiquitylated RNAPII is hypophosphorylated at serine 5 in vivo, and mutation of the serine 5 phosphatase SSU72 inhibits RNAPII degradation. These results identify several mechanisms that confine ubiquitylation of RNAPII to the forms of the enzyme that arrest during elongation.


Expression and modulation of RPE cell membrane complement regulatory proteins.
Complement, inflammation, and oxidant injury contribute to age-related macular degeneration (AMD). Membrane complement regulatory proteins (mCRPs) such as CD46, CD55, and CD59, protect host cells from complement attack. The factors that regulate RPE mCRP expression are not well understood. In this study, the authors sought to determine whether cytokines and hydroquinone (HQ) affect mCRP expression in cultured human RPE (hRPE) and cultured mouse RPE (mRPE) cells. Cultured hRPE and mRPE cells were stimulated with cytokines for various times or with HQ for multiple 6-hour periods. mRNA and protein expression of mCRPs in cultured hRPE cells from 10 donors, native hRPE, and mouse eyecups and native mRPE cells were evaluated by real-time RT-PCR, Western blot analysis, and flow cytometry, respectively. Three mCRPs were expressed in cultured hRPE cells (CD59>CD46>CD55). CD46 and CD59 protein were detected in native hRPE cells. CD59 protein levels in cultured hRPE cells were higher than in native hRPE cells. CD46 protein polymorphisms were observed in cultured hRPE cells. Cultured hRPE cell mCRP expression was upregulated by TNF-alpha, IL-1beta, and a repetitive nonlethal dose of HQ. CD59a levels were higher in mouse eyecups than in nonocular tissues. Mouse mCRP mRNA and protein were detected in native mRPE cells. Responsiveness to cytokines in cultured mRPE cells differed from that in cultured hRPE cells. Human and mouse RPE cell mCRPs are upregulated by inflammatory cytokines and repetitive nonlethal oxidant exposure in a species-specific manner. Increased cell mCRPs may help to protect RPE cells from complement- and oxidant-mediated injury in diseases such as AMD.


The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to the nitrogen source and has homology to glutathione s-transferases.
The URE2 gene of Saccharomyces cerevisiae has been cloned and sequenced. It encodes a predicted polypeptide of 354 amino acids with a molecular weight of 40,226. Deletion of the first 63 amino acids does not have any effect on the function of the protein. Studies with disruption alleles of the URE2 and GLN3 genes showed that both genes regulate GLN1 and GDH2, the structural genes for glutamine synthetase and NAD-linked glutamate dehydrogenase, respectively, at the transcriptional level, but expression of the regulatory genes does not appear to be regulated. Active URE2 gene product was required for the inactivation of glutamine synthetase upon addition of glutamine to cells growing with glutamate as the source of nitrogen. The predicted URE2 gene product has homology to glutathione S-transferases. The gene has been mapped to chromosome XIV, 5.9 map units from petX and 3.4 map units from kex2.


HUM5007, a novel combination of thermogenic compounds, and 3-acetyl-7-oxo-dehydroepiandrosterone: each increases the resting metabolic rate of overweight adults.
This study tested the hypothesis that 3-acetyl-7-oxo-dehydroepiandrosterone alone (7-Keto) and in combination with calcium citrate, green tea extract, ascorbic acid, chromium nicotinate and cholecalciferol (HUM5007) will increase the resting metabolic rate (RMR) of overweight subjects maintained on a calorie-restricted diet. In this randomized, double-blind, placebo-controlled, crossover trial, overweight adults on a calorie-restricted diet were randomized to three 7-day treatment periods with 7-Keto, HUM5007 or placebo. Resting metabolic rate was measured by indirect calorimetry at the beginning and end of each treatment period with a 7-day washout between testing periods. Of 45 subjects enrolled, 40 completed the study (30 women, 10 men; mean age, 38.5 years; mean mass index, 32.0 kg/m(2)). During the placebo treatment, RMR decreased by 3.9% (75+/-111 kcal/day; mean+/-S.D.); however, RMR increased significantly by 1.4% (21+/-115 kcal/day) and 3.4% (59+/-118 kcal/day) during the 7-Keto and HUM5007 treatment periods, respectively (each compared to placebo, P=.001). No significant differences were found between the treatment periods with respect to compliance or adverse events. In this study, the administration of HUM5007 or 7-Keto reversed the decrease in RMR normally associated with dieting. HUM5007 and 7-Keto increased RMR above basal levels and may benefit obese individuals with impaired energy expenditure. HUM5007 and 7-Keto were generally well tolerated and no serious adverse events were reported.


Routine Depression Screenings for Advanced Cancer Patients: Reducing Disparities, Identifying Depression, and Improving Quality of Life.
Advanced cancer patients are at an increased risk of developing depressive symptoms, which can lead to major depressive disorder and a poor quality of life. It is important that symptoms of depression to be addressed early and frequently throughout the trajectory of the disease process. Depression is underdiagnosed and therefore undertreated in advanced cancer patients. Clinicians often fail to perform regular depression screenings as recommended by the National Comprehensive Cancer Network. Depressive symptoms are overlooked as they tend to overlap with the effects of disease progression and cancer treatments. Patients' complaints of anorexia, chronic pain, and sleep disturbances do not necessarily trigger practitioners to perform depression screenings. African Americans with advanced cancer are at a higher risk of developing depression, but may not identify as depressed due to the stigma of mental health in the black community. Screening tools such as the 2- and 9-item Patient Health Questionnaire, Beck Depression Inventory II, Hospital Anxiety and Depression Scale, and the Distress Thermometer and Problem List are common brief instruments that can screen for depression. Providing early symptom relief of depressive symptoms through psychotherapy and pharmacologic interventions will benefit the patient, family, and caregivers while improving the quality of life throughout the trajectory of the illness.


'Only twice a year': a qualitative exploration of 6-month antiretroviral treatment refills in adherence clubs for people living with HIV in Khayelitsha, South Africa.
Longer intervals between routine clinic visits and medication refills are part of patient-centred, differentiated service delivery (DSD). They have been shown to improve patient outcomes as well as optimise health services-vital as 'universal test-and-treat' targets increase numbers of HIV patients on antiretroviral treatment (ART). This qualitative study explored patient, healthcare worker and key informant experiences and perceptions of extending ART refills to 6 months in adherence clubs in Khayelitsha, South Africa. In-depth interviews were conducted in isiXhosa with purposively selected patients and in English with healthcare workers and key informants. All transcripts were audio-recorded, transcribed and translated to English, manually coded and thematically analysed. The participants had been involved in a randomised controlled trial evaluating multi-month ART dispensing in adherence clubs, comparing 6-month and 2-month refills. Twenty-three patients, seven healthcare workers and six key informants. Patients found that 6-month refills increased convenience and reduced unintended disclosure. Contrary to key informant concerns about patients' responsibility to manage larger quantities of ART, patients receiving 6-month refills were highly motivated and did not face challenges transporting, storing or adhering to treatment. All participant groups suggested that strict eligibility criteria were necessary for patients to realise the benefits of extended dispensing intervals. Six-month refills were felt to increase health system efficiency, but there were concerns about whether the existing drug supply system could adapt to 6-month refills on a larger scale. Patients, healthcare workers and key informants found 6-month refills within adherence clubs acceptable and beneficial, but concerns were raised about the reliability of the supply chain to manage extended multi-month dispensing. Stepwise, slow expansion could avoid overstressing supply and allow time for the health system to adapt, permitting 6-month ART refills to enhance current DSD options to be more efficient and patient-centred within current health system constraints.


Shipping augmentative biocontrol agents.
The majority of worldwide trading in live arthropods is done by specialised companies that produce and distribute beneficial insects, mites and nematodes for augmentative biological control of pests on crops. These beneficial arthropods, predators or parasitoids of crop pests and some nuisance species, which are harmless to humans, plants and other animals, are mass-reared, transported and released in the target environment as a viable alternative to chemical pesticides. As such, they play a major role in achieving a more sustainable and regenerative agriculture. Thousands of international air shipments are performed yearly by up to 30 major companies. Over the decades that this industry has been active, no significant transport problems have arisen. This is due to stringent standards that the industry has generated, including designated departments to deal with quality control and logistics, as well as guides for each species concerning packaging, storage and transport conditions. In addition, transporters always adhere to the regulatory standards of the importing and exporting countries, which is attested to by the documents included by the companies with each shipment. Two major issues arise when shipping live arthropods: first, the need to maintain stable environmental conditions within the shipping units (maintaining a cool chain) and, second, minimising transport time (preventing delays). This paper discusses the procedures implemented by the companies to assure quality, the documents required and provided currently, and the challenges to the safe transportation of beneficial organisms.


Is bisphenol S a safe substitute for bisphenol A in terms of metabolic function? An in vitro study.
As bisphenol A (BPA) has been shown to induce adverse effects on human health, especially through the activation of endocrine pathways, it is about to be withdrawn from the European market and replaced by analogues such as bisphenol S (BPS). However, toxicological data on BPS is scarce, and so it is necessary to evaluate the possible effects of this compound on human health. We compared the effect of BPA and BPS on obesity and hepatic steatosis processes using low doses in the same range as those found in the environment. Two in vitro models were used, the adipose cell line 3T3-L1 and HepG2 cells, representative of hepatic functions. We analyzed different parameters such as lipid and glucose uptakes, lipolysis, leptin production and the modulation of genes involved in lipid metabolism and energy balance. BPA and BPS induced an increase in the lipid content in the 3T3-L1 cell line and more moderately in the hepatic cells. We also observed a decrease in lipolysis after bisphenol treatment of adipocytes, but only BPS was involved in the increase in glucose uptake and leptin production. These latter effects could be linked to the modulation of SREBP-1c, PPARγ, aP2 and ERRα and γ genes after exposure to BPA, whereas BPS seems to target the PGC1α and the ERRγ genes. The findings suggest that both BPA and BPS could be involved in obesity and steatosis processes, but through two different metabolic pathways.


The biogeochemical cycles of trace metals in the oceans.
Planktonic uptake of some essential metals results in extraordinarily low concentrations in surface seawater. To sequester or take up these micronutrients, various microorganisms apparently release strong complexing agents and catalyze redox reactions that modify the bioavailability of trace metals and promote their rapid cycling in the upper water column. In turn, the low availability of some metals controls the rate of photosynthesis in parts of the oceans and the transformation and uptake of major nutrients such as nitrogen. The extremely low concentrations of several essential metals are both the cause and the result of ultraefficient uptake systems in the plankton and of widespread replacement of metals by one another for various biochemical functions.


Physical activity, sedentary behavior, and vitamin D metabolites.
Physical activity is associated with circulating 25-hydroxyvitamin D (25(OH)D). However, the influence of activity and/or sedentary behavior on the biologically active, seco-steroid hormone 1α,25-dihydroxyvitamin D (1,25(OH)2D) is unknown. We conducted a cross-sectional analysis among ursodeoxycholic acid (UDCA) randomized trial participants (n=876) to evaluate associations between physical activity, sedentary behavior, and circulating vitamin D metabolite concentrations. Continuous vitamin D metabolite measurements and clinical thresholds were evaluated using multiple linear and logistic regression models, mutually adjusted for either 1,25(OH)2D or 25(OH)D and additional confounding factors. A statistically significant linear association between 1,25(OH)2D and moderate-vigorous physical activity per week was strongest among women (β (95% CI): 3.10 (1.51-6.35)) versus men (β (95% CI): 1.35 (0.79-2.29)) in the highest tertile of activity compared to the lowest (p-interaction=0.003). Furthermore, 25(OH)D was 1.54ng/ml (95% CI 1.09-1.98) higher per hour increase in moderate-vigorous activity (p=0.001) and odds of sufficient 25(OH)D status was higher among physically active participants (p=0.001). Sedentary behavior was not significantly associated with either metabolite in linear regression models, nor was a statistically significant interaction by sex identified. The current study identified novel associations between physical activity and serum 1,25(OH)2D levels, adjusted for 25(OH)D concentrations. These results identify the biologically active form of vitamin D as a potential physiologic mechanism related to observed population-level associations between moderate-vigorous physical activity with bone health and chronic disease risk. However, future longitudinal studies are needed to further evaluate the role of physical activity and vitamin D metabolites in chronic disease prevention.


Hydrogen Bonding and Related Properties in Liquid Water: A Car-Parrinello Molecular Dynamics Simulation Study.
The local hydrogen-bonding structure and dynamics of liquid water have been investigated using the Car-Parrinello molecular dynamics simulation technique. The radial distribution functions and coordination numbers around water molecules have been found to be strongly dependent on the number of hydrogen bonds formed by each molecule, revealing also the existence of local structural heterogeneities in the structure of the liquid. The results obtained have also revealed the strong effect of the local hydrogen-bonding network on the local tetrahedral structure and entropy. The investigation of the dynamics of the local hydrogen-bonding network in liquid water has shown that this network is very labile, and the hydrogen bonds break and reform very rapidly. Nevertheless, it has been found that the hydrogen-bonding states associated with the formation of four hydrogen bonds by a water molecule exhibit the largest survival probability and corresponding lifetime. The reorientational motions of water molecules have also been found to be strongly dependent on their initial hydrogen-bonding state. Finally, the dependence of the librational and vibrational modes of water molecules on the local hydrogen-bonding network has been carefully examined, revealing a significant effect upon the libration and bond-stretching peak frequencies. The calculated low frequency peaks come in agreement with previously reported interpretations of the experimental low-frequency Raman spectrum of liquid water.


PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase.
The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations. PRAS40 inhibits cell growth, S6K1 phosphorylation, and rheb-induced activation of the mTORC1 pathway, and in vitro it prevents the great increase in mTORC1 kinase activity induced by rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which prevents its inhibition of mTORC1 in cells and in vitro. We propose that the relative strengths of the rheb- and PRAS40-mediated inputs to mTORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both.


A comparison of the EuroQol-5D and the Health Utilities Index mark 3 in patients with rheumatic disease.
To compare the performance of 2 commonly used utility-based health-related quality of life (HRQoL) instruments [the EuroQol-5D (EQ-5D) and Health Utilities Index mark 3 (HUI3)] in patients with rheumatic disease. Consecutive outpatients with rheumatic diseases were interviewed twice within 2 weeks using a standard questionnaire containing the EQ-5D, HUI3, and the Medical Outcome Study Short-Form 36 Health Survey (SF-36, used to categorize health status) and assessing clinical and demographic characteristics. EQ-5D and HUI3 utility scores were compared and their construct validity and test-retest reliability were examined by comparing these scores in groups differing in health status and using intraclass correlation coefficients (ICC), respectively. EQ-5D and HUI3 utility scores in 114 patients differentiated well between varying health states; e.g., patients with higher SF-36 vitality scores had better EQ-5D and HUI3 utility scores (mean: 0.79 for both instruments) than patients with lower vitality scores (mean: 0.68 and 0.69, respectively) (p < 0.01 for both comparisons). ICC values for the EQ-5D and HUI3 were 0.64 and 0.75, respectively (n = 90, median interval: 7 days). EQ-5D and HUI3 utility scores were similar (mean +/- SD: 0.75 +/- 0.21 vs 0.76 +/- 0.17, p = 0.647, paired t test) and showed moderate correlation (Spearman's r: 0.45, p < 0.001). Differences were present in patients' responses to these 2 instruments: e.g., 12 patients reporting no problems with mobility (EQ-5D item) reported different levels of disability with ambulation (HUI3 item). The EQ-5D and HUI3 performed equally well in measuring utility-based HRQoL in patients with rheumatic disease, although they measured slightly different, though related, dimensions of health.


John's $12 tonic: press coverage of the government's selling of a private health insurance rebate.
To document representations of the 1998 introduction of a 30% rebate on private health insurance in the three most-read daily Sydney newspapers. Thematic frame analysis of 131 newspaper articles. The rebate was opposed through two frames: that it was ineffective and unfair, and that it was politically motivated. Four supportive frames were more complex: the rebate was justified by claims that public health care was collapsing, that responsible citizens should pay for their own health care, and that individuals would benefit financially. There was also a focus on the political battle in the Senate. The newspaper with the readership least likely to benefit from the rebate supported it most strongly. Framing was strongly episodic (two dimensional, decontextualised and case-study based), limiting political accountability, and the anti-rebate case was presented less memorably. Community action around the issue was not encouraged, individual responsibility was emphasised and universal health care was not promoted as fair or necessary. Different readerships received different messages about the rebate. There is an urgent need to promote the value of the public health care system and make the future of Medicare compelling for news editors and the public.


A distinct set of Drosophila brain neurons required for neurofibromatosis type 1-dependent learning and memory.
Nonspecific cognitive impairments are one of the many manifestations of neurofibromatosis type 1 (NF1). A learning phenotype is also present in Drosophila melanogaster that lack a functional neurofibromin gene (nf1). Multiple studies have indicated that Nf1-dependent learning in Drosophila involves the cAMP pathway, including the demonstration of a genetic interaction between Nf1 and the rutabaga-encoded adenylyl cyclase (Rut-AC). Olfactory classical conditioning experiments have previously demonstrated a requirement for Rut-AC activity and downstream cAMP pathway signaling in neurons of the mushroom bodies. However, Nf1 expression in adult mushroom body neurons has not been observed. Here, we address this discrepancy by demonstrating (1) that Rut-AC is required for the acquisition and stability of olfactory memories, whereas Nf1 is only required for acquisition, (2) that expression of nf1 RNA can be detected in the cell bodies of mushroom body neurons, and (3) that expression of an nf1 transgene only in the alpha/beta subset of mushroom body neurons is sufficient to restore both protein synthesis-independent and protein synthesis-dependent memory. Our observations indicate that memory-related functions of Rut-AC are both Nf1-dependent and -independent, that Nf1 mediates the formation of two distinct memory components within a single neuron population, and that our understanding of Nf1 function in memory processes may be dissected from its role in other brain functions by specifically studying the alpha/beta mushroom body neurons.


Time course and host responses to Escherichia coli urinary tract infection in genetically distinct mouse strains.
Recurrent urinary tract infections (UTIs) are a significant clinical problem for many women; however, host susceptibility factors have not been completely defined. The mouse model of induced UTI provides an experimental environment in which to identify specific host characteristics that are important in initial bacterial colonization of the urinary tract and in resolution of an infection. This study examined initial susceptibility, bacterial clearance, and host defense mechanisms during induction and resolution of Escherichia coli UTIs in genetically distinct strains of mice. Of the ten inbred strains tested, six (BALB/c, C3H/HeN, C57BL/6, DBA.1, DBA.2, and AKR) showed progressive resolution of bladder infections over a 14-day period. A constant, low-level bladder infection was observed in SWR and SJL mice. High bladder infection levels persisted over the 14-day study period in C3H/HeJ and C3H/OuJ mice. Kidney infection levels generally correlated with bladder infection levels, especially in C3H/HeJ and C3H/OuJ mice, the two most susceptible strains, in which infections became more severe with time after challenge. The degree of inflammation in bladder and kidneys, as well as antibody-forming cell responses, positively correlated with infection intensity in all strains except C3H/HeJ, which had minimal inflammation despite high infection levels. These results demonstrate two important aspects of host defense against UTI. First, the innate immune response to an infection in the bladder or kidneys consists primarily of local inflammation, which is followed by an adaptive response characterized in part by an antibody response to the infecting bacteria. Second, a UTI will be spontaneously resolved in most cases; however, in mice with specific genetic backgrounds, a UTI can persist for an extended length of time. The latter result strongly suggests that the presence or absence of specific host genes will determine how effectively an E. coli UTI will be resolved.


AAV1/2-mediated CNS gene delivery of dominant-negative CCL2 mutant suppresses gliosis, beta-amyloidosis, and learning impairment of APP/PS1 mice.
Accumulation of aggregated amyloid-beta (Abeta) peptide was studied as an initial step for Alzheimer's disease (AD) pathogenesis. Following amyloid plaque formation, reactive microglia and astrocytes accumulate around plaques and cause neuroinflammation. Here brain chemokines play a major role for the glial accumulation. We have previously shown that transgenic overexpression of chemokine CCL2 in the brain results in increased microglial accumulation and diffuse amyloid plaque deposition in a transgenic mouse model of AD expressing Swedish amyloid precursor protein (APP) mutant. Here, we report that adeno-associated virus (AAV) serotype 1 and 2 hybrid efficiently deliver 7ND gene, a dominant-negative CCL2 mutant, in a dose-response manner and express >1,000-fold higher recombinant CCL2 than basal levels after a single administration. AAV1/2 hybrid virus principally infected neurons without neuroinflammation with sustained expression for 6-months. 7ND expressed in APP/presenilin-1 (APP/PS1) bigenic mice reduced astro/microgliosis, beta-amyloidosis, including suppression of both fibrillar and oligomer Abeta accumulation, and improved spatial learning. Our data support the idea that the AAV1/2 system is a useful tool for CNS gene delivery, and suppression of CCL2 may be a therapeutic target for the amelioration of AD-related neuroinflammation.


Frog Prince transposon-based RNAi vectors mediate efficient gene knockdown in human cells.
We have developed a stable RNA interference (RNAi) delivery system that is based on the Frog Prince transposable element. This plasmid-based vector system combines the gene silencing capabilities of H1 polymerase III promoter-driven short hairpin RNAs (shRNA) with the advantages of stable and efficient genomic integration of the shRNA cassette mediated by transposition. We show that the Frog Prince-based shRNA expressing system can efficiently knock down the expression of both exogenous as well as endogenous genes in human cells. Furthermore, we use the Frog Prince-based system to study the effect of knockdown of the DNA repair factor Ku70 on transposition of the Sleeping Beauty transposon. Transposon-mediated genomic integration ensures that the shRNA expression cassette and a selectable marker gene within the transposon remain intact and physically linked. We demonstrate that a major advantage of our vector system over plasmid-based shRNA delivery is both its enhanced frequency of intact genomic integration as well as higher target suppression in transgenic human cells. Due to its simplicity and effectiveness, transposon-based RNAi is an emerging tool to facilitate analysis of gene function through the establishment of stable loss-of-function cell lines.


Functioning in lymphedema from the patients' perspective using the International Classification of Functioning, Disability and health (ICF) as a reference.
To identify and quantify meaningful concepts in lymphedema from the patients' perspectives using the International Classification of Functioning, Disability and Health (ICF). Six focus group interviews in five different centers were organized, audiotaped, transcribed verbatim and analyzed. A total of 2681 relevant ICF linkings were performed with the focus group data, resulting in 130 different second-level categories. Of these 130 second-level categories, 41 (31.5%) categories were categorized as Body Functions, 20 (15.5%) as Body Structures, 41 (31.5%) as Activities and Participation, and 28 (21.5%) as Environmental Factors. Overall, the most important issues according to the patients were the use of hosiery and bandages, support and relationships, and the shape of structures related to movement. Based on their experiences with lymphedema, patients reported activity limitations and participation restrictions combined with impaired body functioning. Anatomical changes (Body Structures) were also often mentioned as a problem in daily life. Environmental factors may act as a barrier or facilitator for patient functioning. The ICF provides a valuable reference to identify concepts in statements from lymphedema patients. The results of this research will be used in the development of ICF Core Sets for lymphedema.


[Paternity and health services. Qualitative research on men's experiences during pregnancy, delivery and postpartum of theirs partners].
Little research has been carried out with regards to the inclusion of men during the birth process. The objective of this paper involves exploring the needs and expectations of the health services manifested by a group of fathers as a result of their experience during the birth process. Qualitative research was carried out in Granada in 2004 via individual interviews with fathers who showed shared responsibility in the upbringing. The profile is: employment, medium-high educational level, one or more child: 0-6 months of age. The transcript was subsequently submitted to hermeneutic analysis. Some semantic constructs are: 1) Health Services do not concede the women as protagonists, 2) Birth process is depending on the body. Fathers can only support and fight for the relevance of men, 3) Men seem like "invisible", 4) Health services inhibit their participation, and 5) have dealings with fathers according to their gender roles. The participants address the relationship between expectations of care during the birth process and unsatisfied demands, and the manner in which they employ the obstacles encountered within health services that inhibit their participation as arguments that confirm their separation from the process. This paper draws attention to the limited scope of the provision of healthcare during the birth process in terms of protagonism afforded to fathers. Indeed, despite their requisitory discourse, the interviewees manifest contradictory attitudes in the face of changes that require them to make commitments. We identify elements that could be improved to adapt services to the needs of fathers and vice versa.


3D printing of hierarchical porous biomimetic hydroxyapatite scaffolds: Adding concavities to the convex filaments.
Porosity plays a key role on the osteogenic performance of bone scaffolds. Direct Ink Writing (DIW) allows the design of customized synthetic bone grafts with patient-specific architecture and controlled macroporosity. Being an extrusion-based technique, the scaffolds obtained are formed by arrays of cylindrical filaments, and therefore have convex surfaces. This may represent a serious limitation, as the role of surface curvature and more specifically the stimulating role of concave surfaces in osteoinduction and bone growth has been recently highlighted. Hence the need to design strategies that allow the introduction of concave pores in DIW scaffolds. In the current study, we propose to add gelatin microspheres as a sacrificial material in a self-setting calcium phosphate ink. Neither the phase transformation responsible for the hardening of the scaffold nor the formation of characteristic network of needle-like hydroxyapatite crystals was affected by the addition of gelatin microspheres. The partial dissolution of the gelatin resulted in the creation of spherical pores throughout the filaments and exposed on the surface, increasing filament porosity from 0.2 % to 67.9 %. Moreover, the presence of retained gelatin proved to have a significant effect on the mechanical properties, reducing the strength but simultaneously giving the scaffolds an elastic behavior, despite the high content of ceramic as a continuous phase. Notwithstanding the inherent difficulty of in vitro cultures with this highly reactive material an enhancement of MG-63 cell proliferation, as well as better spreading of hMSCs was recorded on the developed scaffolds. STATEMENT OF SIGNIFICANCE: Recent studies have stressed the role that concave surfaces play in tissue regeneration and, more specifically, in osteoinduction and osteogenesis. Direct ink writing enables the production of patient-specific bone grafts with controlled architecture. However, besides many advantages, it has the serious limitation that the surfaces obtained are convex. In this article, for the first time we develop a strategy to introduce concave pores in the printed filaments of biomimetic hydroxyapatite by incorporation and partial dissolution of gelatin microspheres. The retention of part of the gelatin results in a more elastic behavior compared to the brittleness of hydroxyapatite scaffolds, while the needle-shaped nanostructure of biomimetic hydroxyapatite is maintained and gelatin-coated concave pores on the surface of the filaments enhance cell spreading.


Chlamydia pneumoniae detection in atherosclerotic plaques in Italy.
Italian investigations have shown an association between Chlamydia pneumoniae infection and atherosclerosis. With the use of several diagnostic techniques, including serology, a microimmunofluorescence test, and nucleic acid amplification methods, a temporal association was found between acute C. pneumoniae reinfection and acute myocardial infarction, suggesting that an acute infection superimposed on a chronic or latent infection may trigger the onset of acute myocardial infarction. C. pneumoniae but not Helicobacter pylori or Mycoplasma pneumoniae was found in atherosclerotic plaques of abdominal aortic aneurysms and the carotid artery. A reverse transcriptase-polymerase chain reaction process confirmed the presence of viable C. pneumoniae in carotid atheromas. Nucleic amplification of peripheral blood mononuclear cells may enable the identification of subjects carrying C. pneumoniae in the vascular wall. Macrolide treatment reduced fibrinogen and C-reactive protein plasma levels and C. pneumoniae burden in patients with atherosclerotic diseases.


A modular planar robotic manipulandum with end-point torque control.
Robotic manipulanda are extensively used in investigation of the motor control of human arm movements. They permit the application of translational forces to the arm based on its state and can be used to probe issues ranging from mechanisms of neural control to biomechanics. However, most current designs are optimized for studying either motor learning or stiffness. Even fewer include end-point torque control which is important for the simulation of objects and the study of tool use. Here we describe a modular, general purpose, two-dimensional planar manipulandum (vBOT) primarily optimized for dynamic learning paradigms. It employs a carbon fibre arm arranged as a parallelogram which is driven by motors via timing pulleys. The design minimizes the intrinsic dynamics of the manipulandum without active compensation. A novel variant of the design (WristBOT) can apply torques at the handle using an add-on cable drive mechanism. In a second variant (StiffBOT) a more rigid arm can be substituted and zero backlash belts can be used, making the StiffBOT more suitable for the study of stiffness. The three variants can be used with custom built display rigs, mounting, and air tables. We investigated the performance of the vBOT and its variants in terms of effective end-point mass, viscosity and stiffness. Finally we present an object manipulation task using the WristBOT. This demonstrates that subjects can perceive the orientation of the principal axis of an object based on haptic feedback arising from its rotational dynamics.


Long-term outcomes of catheter ablation of atrial fibrillation: a systematic review and meta-analysis.
In the past decade, catheter ablation has become an established therapy for symptomatic atrial fibrillation (AF). Until very recently, few data have been available to guide the clinical community on the outcomes of AF ablation at ≥3 years of follow-up. We aimed to systematically review the medical literature to evaluate the long-term outcomes of AF ablation. A structured electronic database search (PubMed, Embase, Web of Science, Cochrane) of the scientific literature was performed for studies describing outcomes at ≥3 years after AF ablation, with a mean follow-up of ≥24 months after the index procedure. The following data were extracted: (1) single-procedure success, (2) multiple-procedure success, and (3) requirement for repeat procedures. Data were extracted from 19 studies, including 6167 patients undergoing AF ablation. Single-procedure freedom from atrial arrhythmia at long-term follow-up was 53.1% (95% CI 46.2% to 60.0%) overall, 54.1% (95% CI 44.4% to 63.4%) in paroxysmal AF, and 41.8% (95% CI 25.2% to 60.5%) in nonparoxysmal AF. Substantial heterogeneity (I(2)>50%) was noted for single-procedure outcomes. With multiple procedures, the long-term success rate was 79.8% (95% CI 75.0% to 83.8%) overall, with significant heterogeneity (I(2)>50%).The average number of procedures per patient was 1.51 (95% CI 1.36 to 1.67). Catheter ablation is an effective and durable long-term therapeutic strategy for some AF patients. Although significant heterogeneity is seen with single procedures, long-term freedom from atrial arrhythmia can be achieved in some patients, but multiple procedures may be required.


Kidney and liver transplants from donors after cardiac death: initial experience at the London Health Sciences Centre.
The disparity between the number of patients waiting for an organ transplant and availability of donor organs increases each year in Canada. Donation after cardiac death (DCD), following withdrawal of life support in patients with hopeless prognoses, is a means of addressing the shortage with the potential to increase the number of transplantable organs. We conducted a retrospective, single-centre chart review of organs donated after cardiac death to the Multi-Organ Transplant Program at the London Health Sciences Centre between July 2006 and December 2007. In total, 34 solid organs (24 kidneys and 10 livers) were procured from 12 DCD donors. The mean age of the donors was 38 (range 18-59) years. The causes of death were craniocerebral trauma (n = 7), cerebrovascular accident (n = 4) and cerebral hypoxia (n = 1). All 10 livers were transplanted at our centre, as were 14 of the 24 kidneys; 10 kidneys were transplanted at other centres. The mean renal cold ischemia time was 6 (range 3-9.5) hours. Twelve of the 14 kidney recipients (86%) experienced delayed graft function, but all kidneys regained function. After 1-year follow-up, kidney function was good, with a mean serum creatinine level of 145 (range 107-220) micromol/L and a mean estimated creatinine clearance of 64 (range 41-96) mL/min. The mean liver cold ischemia time was 5.8 (range 5.5-8) hours. There was 1 case of primary nonfunction requiring retransplantation. The remaining 9 livers functioned well. One patient developed a biliary anastomotic stricture that resolved after endoscopic stenting. All liver recipients were alive after a mean follow-up of 11 (range 3-20) months. Since the inception of this DCD program, the number of donors referred to our centre has increased by 14%. Our initial results compare favourably with those from the transplantation of organs procured from donors after brain death. Donation after cardiac death can be an important means of increasing the number of organs available for transplant, and its widespread implementation in Canada should be encouraged.


Implementation of a Medical Education Rotation for Senior Emergency Medicine Residents.
This medical education (MedEd) rotation is designed for post graduate year 3 (PGY-3) residents. The rotation runs over one month for each PGY-3. Resident physicians have teaching responsibilities during and after training. These responsibilities expand beyond teaching medical students and junior residents to include teaching advanced practice providers, nursing colleagues, and prehospital personnel.1 The need for formal teaching curricula in graduate medical education is recognized, but practical examples are lacking.2. Our objectives were to provide our senior residents with exposure to various aspects of the field of MedEd, to further develop their teaching skills and to encourage them to consider a career in academic emergency medicine. The educational strategies used in this curriculum include: 1) clinical shifts supervising small groups of medical students with dedicated faculty supervision, 2) a structured simulation-based medical student teaching activity where the resident is able to provide feedback and teach medical students, 3) a MedEd project, 4) required readings that cover a variety of topics including education theory, curriculum design, and feedback, 5) case-based didactic presentation at our monthly case conference, and 6) one hour of postgraduate year 1 (PGY-1) small group facilitation focusing on fundamentals of emergency medicine. PGY-3 residents completed an online survey prior to residency graduation. The timing of the survey was purposefully delayed to the end of the academic year to allow the residents time to practice techniques they learned during their MedEd rotation. Thirteen residents (93%) completed a survey. Five residents (38%) reported that the rotation had "some" or more impact on their career decision. The other 8 residents reported "almost no impact" or "a little bit of impact." Ten residents (77%) reported that they would "sometimes," "often," or "almost always" use the teaching techniques they learned during the rotation. The highest rated activities were simulation-based teaching and dedicated clinical teaching shifts. Confidence with bedside teaching improved after the session, with a median confidence before the session of 3/5 (moderately confident; IQR 2-3) and a median confidence after the session of 4/5 (quite confident; IQR 3-4, p=0.006). Our MedEd rotation improved teaching confidence but had low impact on career decision. Residents rated the interactive, faculty-supervised components of the rotation highest. We recommend that programs interested in instituting a MedEd rotation first trial the rotation as an elective and utilize established formal teaching activities. Medical Education, resident physician, medical student teaching, simulation, academic medicine.


Mechanisms of tamoxifen-induced apoptosis.
Tamoxifen (TAM) has been used in the treatment of breast cancer for over a decade. The observed clinical efficacy of TAM has been attributed to both growth arrest and induction of apoptosis within the breast cancer cells. Although the primary mechanism of action of TAM is believed to be through the inhibition of estrogen receptor (ER), research over the years has indicated that additional, non-ER-mediated mechanisms exist. These include modulation of signaling proteins such as protein kinase C (PKC), calmodulin, transforming growth factor-beta (TGFbeta), and the protooncogene c-myc. Recent studies, including those from our laboratory, have implicated the role of caspases and mitogen-activated protein kinases (MAPK), including c-Jun N-terminal kinase (JNK) and p38 in TAM-induced apoptotic signaling. Oxidative stress, mitochondrial permeability transition (MPT), ceramide generation as well as changes in cell membrane fluidity may also play important roles in TAM-induced apoptosis. These various signaling pathways underlying TAM-induced apoptosis will be reviewed in this article.


Micropatterned immobilization of a G protein-coupled receptor and direct detection of G protein activation.
G protein-coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined orientation to substrates. By surface plasmon resonance (SPR), we were able to follow ligand binding, G protein activation, and receptor deactivation of a representative GPCR, bovine rhodopsin. Microcontact printing was used to produce micrometer-sized patterns with high contrast in receptor activity. These patterns can be used for local referencing to enhance the sensitivity of chip-based assays. The immobilized receptor was stable both for hours and during several activation cycles. A ligand dose-response curve with the photoactivatable agonist 11-cis-retinal showed a half-maximal signal at 120 nM. Our findings may be useful to develop novel assay formats for GPCRs based on receptor immobilization to solid supports, particularly to sensor surfaces.


Effects of ozonated autohemotherapy on the antioxidant capacity of Thoroughbred horses.
The performance of horses undergoing regular intense exercise is adversely affected by oxidative stress. Thus, it is important to increase antioxidant production in horses in order to reduce oxidative stress. Ozonated autohemotherapy (OAHT) reportedly promotes antioxidant production. This study aimed to evaluate the effects of OAHT on antioxidant capacity. Ten Thoroughbred horses were used in this study. After the OAHT, we collected serum samples and measured biological antioxidant potential (BAP). We found that BAP began to increase after the OAHT and was significantly higher in the OAHT group than at 3 (P<0.01) and 7 days (P<0.05) after OAHT than in the control group at 3 and 7 days after starting collection of blood samples. Therefore, it was shown that OAHT improved the antioxidant capacity of the horses.


Auditory hair cell centrioles undergo confined Brownian motion throughout the developmental migration of the kinocilium.
Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements of the kinocilium basal body (mother centriole) and its daughter centriole, which we analyzed using particle tracking and modeling. We found that both hair cell centrioles undergo confined Brownian movements around their equilibrium positions, under the apparent constraint of a radial restoring force of ∼0.1 pN. This magnitude depended little on centriole position, suggesting nonlinear interactions with constraining, presumably cytoskeletal elements. The only dynamic change observed during the period of kinocilium migration was a doubling of the centrioles' confinement area taking place early in the process. It emerges from these static and dynamic observations that kinocilia migrate gradually in parallel with the organization of hair cells into rows during cochlear neuroepithelium extension. Analysis of the confined motion of hair cell centrioles under normal and pathological conditions should help determine which structures contribute to the restoring force exerting on them.


Developing and validating risk prediction models in an individual participant data meta-analysis.
Risk prediction models estimate the risk of developing future outcomes for individuals based on one or more underlying characteristics (predictors). We review how researchers develop and validate risk prediction models within an individual participant data (IPD) meta-analysis, in order to assess the feasibility and conduct of the approach. A qualitative review of the aims, methodology, and reporting in 15 articles that developed a risk prediction model using IPD from multiple studies. The IPD approach offers many opportunities but methodological challenges exist, including: unavailability of requested IPD, missing patient data and predictors, and between-study heterogeneity in methods of measurement, outcome definitions and predictor effects. Most articles develop their model using IPD from all available studies and perform only an internal validation (on the same set of data). Ten of the 15 articles did not allow for any study differences in baseline risk (intercepts), potentially limiting their model's applicability and performance in some populations. Only two articles used external validation (on different data), including a novel method which develops the model on all but one of the IPD studies, tests performance in the excluded study, and repeats by rotating the omitted study. An IPD meta-analysis offers unique opportunities for risk prediction research. Researchers can make more of this by allowing separate model intercept terms for each study (population) to improve generalisability, and by using 'internal-external cross-validation' to simultaneously develop and validate their model. Methodological challenges can be reduced by prospectively planned collaborations that share IPD for risk prediction.


Valproic Acid Combined with Zoledronate Enhance γδ T Cell-Mediated Cytotoxicity against Osteosarcoma Cells via the Accumulation of Mevalonate Pathway Intermediates.
The long-term survival of osteosarcoma has remained unchanged in the last several decades. Immunotherapy is proved to be a promising therapeutic strategy against osteosarcoma, especially for those with metastasis. Our previous study explored the sensibilization of zoledronate (ZOL) in γδ T cell-mediated cytotoxicity against osteosarcoma, but we have not yet elucidated the specific mechanism. Besides, high concentration is required to achieve these effects, whereas plasma ZOL concentration declines rapidly in the circulation. Valproic acid (VPA), a histone deacetylase inhibitor commonly used as the antiepileptic drug, has attracted much attention due to its synergistic antitumor efficacy with chemotherapy or immunotherapy. Here, we demonstrated that VPA combined with ZOL revealed the synergistic effect in enhancing antitumor efficacy of γδ T cells against osteosarcoma cells. This enhancement was mainly TCR-mediated and largely dependent on granule exocytose pathway. Of note, our findings indicated that ZOL sensitized osteosarcoma cells to γδ T cells by increasing the accumulation of the mevalonate pathway intermediates, which could be facilitated by VPA. We also found that this combination had similar effects on primary osteosarcoma cells. All the results suggested that VPA combined with ZOL could reduce the dose required to achieve a significant antitumor effect of γδ T cells, promoting it to be a novel therapy against osteosarcoma.


Calibration of (109)Cd KXRF systems for in vivo bone lead measurements: the guiding role of the assumptions for least-squares regression in practical problem solving.
The use of least-squares regression to probe the level of lead contamination of plaster of Paris standards in the calibration of (109)Cd KXRF systems for bone lead measurement, as well as the use of iteratively reweighted least-squares (IRLS) in the case of violation of the assumptions for ordinary least-squares (OLS), is discussed here. One common violation is non-uniform residual variance, which makes the use of OLS inappropriate due to strong influence of points with large variance on the calibration line and variance of the slope and intercept. Comparison between OLS and IRLS in that case showed that IRLS estimates of the intercept are significantly smaller and more precise than OLS estimates, while a less marked increase in the calibration slope is observed when IRLS is used. Moreover, OLS underestimates bone lead concentrations at low levels of lead exposure and overestimates those concentrations at higher levels. These discrepancies are smaller in magnitude than the measurement uncertainty of conventional systems, except for high concentrations. For the newly developed cloverleaf systems, the suggested differences at bone lead concentrations below 17 ppm are comparable to the minimum detection limit, but are larger than the measurement uncertainty for bone lead concentrations above 60 ppm.


[Analysis of the Cochrane Review: Chromoscopy Versus Conventional Endoscopy for the Detection of Polyps in the Colon and Rectum. Cochrane Database Syst Rev. 2016;4:CD006439].
Although colonoscopy is considered the most accurate test for the investigation of colorectal polyps, lesions ≤ 10 mm may be missed in approximately 10%. Chromoscopy may increase the detection of colorectal polyps. A systematic review and meta-analysis was performed to investigate the benefit of cromoscopy in colorectal polyps detection. Seven randomized controlled trials were included (incuding 2,727 participants) comparing polyp detection (both neoplastic and non- neoplastic polyps) in patients submitted to conventional colonoscopy or colonoscopy with chromoscopy. Chromoscopy was associated with a significant improvement in all detection endpoints (number of polyps, number of neoplastic polyps, number of diminutive polyps, number of neoplastic diminutive polyps, proportion of patients with at least one polyp and proportion of patients with at least one neoplastic polyp). However, when highdefinition colonoscopes were used in the control group, chromoscopy benefit does not seem to be significant. Besides, routine use of chromoscopy may have implications in the accessibility of colonoscopy and in the effectiveness of screening programs. Moreover, the benefit of chromoscopy in the era of high-definition colonoscopy is questionable.


Climate change and food security: health impacts in developed countries.
Anthropogenic climate change will affect global food production, with uncertain consequences for human health in developed countries. We investigated the potential impact of climate change on food security (nutrition and food safety) and the implications for human health in developed countries. Expert input and structured literature searches were conducted and synthesized to produce overall assessments of the likely impacts of climate change on global food production and recommendations for future research and policy changes. Increasing food prices may lower the nutritional quality of dietary intakes, exacerbate obesity, and amplify health inequalities. Altered conditions for food production may result in emerging pathogens, new crop and livestock species, and altered use of pesticides and veterinary medicines, and affect the main transfer mechanisms through which contaminants move from the environment into food. All these have implications for food safety and the nutritional content of food. Climate change mitigation may increase consumption of foods whose production reduces greenhouse gas emissions. Impacts may include reduced red meat consumption (with positive effects on saturated fat, but negative impacts on zinc and iron intake) and reduced winter fruit and vegetable consumption. Developed countries have complex structures in place that may be used to adapt to the food safety consequences of climate change, although their effectiveness will vary between countries, and the ability to respond to nutritional challenges is less certain. Climate change will have notable impacts upon nutrition and food safety in developed countries, but further research is necessary to accurately quantify these impacts. Uncertainty about future impacts, coupled with evidence that climate change may lead to more variable food quality, emphasizes the need to maintain and strengthen existing structures and policies to regulate food production, monitor food quality and safety, and respond to nutritional and safety issues that arise.


Is there a need for autopsies in the management of fungal disease?
The autopsy rates in Germany became low like in other European, American and Asian countries. Main reasons for this development are the lack of acceptance of autopsy in the society as well as in the medical profession, the introduction of a requirement for consent, unclear legal position, the public health system, pressure of costs and a change in the field of activity in pathology with much more diagnostics of surgical and biopsy material. The autopsy is missing with respect to the reliability of causes of death and morbidity statistics and other epidemiological studies. Published data indicate that up to 20-30% of patients who die in hospitals have important diseases/lesions that remain undetected before death but that are found at autopsy. For infectious diseases, the data are similar. Therefore, a higher incidence of invasive fungal infections was found. Some rare fungal disorders are diagnosed by autopsy. Only exact death statistics makes specific health care possible and is cost saving in a public health system in the long term. Autopsy remains an important tool for quality control in medical diagnostic and therapeutic activity. It is also essential for fundamental medical education and further training.


Interrelated effects of mycorrhiza and free-living nitrogen fixers cascade up to aboveground herbivores.
Aboveground plant performance is strongly influenced by belowground microorganisms, some of which are pathogenic and have negative effects, while others, such as nitrogen-fixing bacteria and arbuscular mycorrhizal fungi, usually have positive effects. Recent research revealed that belowground interactions between plants and functionally distinct groups of microorganisms cascade up to aboveground plant associates such as herbivores and their natural enemies. However, while functionally distinct belowground microorganisms commonly co-occur in the rhizosphere, their combined effects, and relative contributions, respectively, on performance of aboveground plant-associated organisms are virtually unexplored. Here, we scrutinized and disentangled the effects of free-living nitrogen-fixing (diazotrophic) bacteria Azotobacter chroococcum (DB) and arbuscular mycorrhizal fungi Glomus mosseae (AMF) on host plant choice and reproduction of the herbivorous two-spotted spider mite Tetranychus urticae on common bean plants Phaseolus vulgaris. Additionally, we assessed plant growth, and AMF and DB occurrence and density as affected by each other. Both AMF alone and DB alone increased spider mite reproduction to similar levels, as compared to the control, and exerted additive effects under co-occurrence. These effects were similarly apparent in host plant choice, that is, the mites preferred leaves from plants with both AMF and DB to plants with AMF or DB to plants grown without AMF and DB. DB, which also act as AMF helper bacteria, enhanced root colonization by AMF, whereas AMF did not affect DB abundance. AMF but not DB increased growth of reproductive plant tissue and seed production, respectively. Both AMF and DB increased the biomass of vegetative aboveground plant tissue. Our study breaks new ground in multitrophic belowground-aboveground research by providing first insights into the fitness implications of plant-mediated interactions between interrelated belowground fungi-bacteria and aboveground herbivores.


Optimal temperature for malaria transmission is dramatically lower than previously predicted.
The ecology of mosquito vectors and malaria parasites affect the incidence, seasonal transmission and geographical range of malaria. Most malaria models to date assume constant or linear responses of mosquito and parasite life-history traits to temperature, predicting optimal transmission at 31 °C. These models are at odds with field observations of transmission dating back nearly a century. We build a model with more realistic ecological assumptions about the thermal physiology of insects. Our model, which includes empirically derived nonlinear thermal responses, predicts optimal malaria transmission at 25 °C (6 °C lower than previous models). Moreover, the model predicts that transmission decreases dramatically at temperatures > 28 °C, altering predictions about how climate change will affect malaria. A large data set on malaria transmission risk in Africa validates both the 25 °C optimum and the decline above 28 °C. Using these more accurate nonlinear thermal-response models will aid in understanding the effects of current and future temperature regimes on disease transmission.


The alleged distinction between euthanasia and the withdrawal of life-sustaining treatment: conceptually incoherent and impossible to maintain.
Richard Epstein, in his book Mortal Peril, supports euthanasia and assisted suicide and rejects the distinction between them and withdrawal treatment. In this essay, Professor Orentlicher argues that Epstein is correct in finding no meaningful moral distinction between euthanasia and treatment withdrawal, examines the reasons why the distinction has persisted in American jurisprudence, and explains why the distinction has eroded. Epstein also concludes in his book that there is no constitutional right to euthanasia or assisted suicide. Professor Orentlicher's response is that constitutionality is not the appropriate inquiry; rather, the better question is whether to recognize a right to assisted suicide once a right to euthanasia in the form of terminal sedation already exists. He answers this question in the affirmative, arguing that assisted suicide enhances patient welfare and reduces risks of abuse in a world with euthanasia.


A national evaluation of the effect of trauma-center care on mortality.
Hospitals have difficulty justifying the expense of maintaining trauma centers without strong evidence of their effectiveness. To address this gap, we examined differences in mortality between level 1 trauma centers and hospitals without a trauma center (non-trauma centers). Mortality outcomes were compared among patients treated in 18 hospitals with a level 1 trauma center and 51 hospitals non-trauma centers located in 14 states. Patients 18 to 84 years old with a moderate-to-severe injury were eligible. Complete data were obtained for 1104 patients who died in the hospital and 4087 patients who were discharged alive. We used propensity-score weighting to adjust for observable differences between patients treated at trauma centers and those treated at non-trauma centers. After adjustment for differences in the case mix, the in-hospital mortality rate was significantly lower at trauma centers than at non-trauma centers (7.6 percent vs. 9.5 percent; relative risk, 0.80; 95 percent confidence interval, 0.66 to 0.98), as was the one-year mortality rate (10.4 percent vs. 13.8 percent; relative risk, 0.75; 95 percent confidence interval, 0.60 to 0.95). The effects of treatment at a trauma center varied according to the severity of injury, with evidence to suggest that differences in mortality rates were primarily confined to patients with more severe injuries. Our findings show that the risk of death is significantly lower when care is provided in a trauma center than in a non-trauma center and argue for continued efforts at regionalization.


Pyrrolyl-, 2-(2-thienyl)pyrrolyl- and 2,5-bis(2-thienyl)pyrrolyl-nucleosides: synthesis, molecular and electronic structure, and redox behaviour of C5-thymidine derivatives.
A series of modified nucleosides based on thymidine have been prepared by Pd-catalysed cross-coupling between N-alkyl-alkynyl functionalised pyrrolyl- (py), 2-(2-thienyl)pyrrolyl- (tp) or 2,5-bis(2-thienyl)pyrrolyl (tpt) groups with 5-iodo-2'-deoxyuridine. The length of the alkyl chain linking the nucleoside and pyrrolyl-containing unit, N(CH(2))(n)C[triple bond, length as m-dash]C-nucleoside (where n = 1-3) was also varied. The compounds have been characterised by (1)H NMR, ES-MS, UV-vis, cyclic voltammetry (CV) and, in some cases, single-crystal X-ray diffraction. Cyclic voltammetry studies demonstrated that all the py-, tp- and tpt-alkynyl derivatives 1-7 can be electrochemically polymerised to form conductive materials. It was found that increasing the N-alkyl chain length in these cases resulted in only minor changes in the oxidation potential. The same behaviour was observed for the tp- and tpt-modified nucleosides 9-12; however, the py-derivative, 8, produced a poorly conducting material. DFT calculations on the one-electron oxidised cation of the modified nucleosides bearing tp or tpt showed that spin density is located on the pyrrolyl and thienyl units in all cases and that the coplanarity of adjacent rings increases upon oxidation. In contrast, in the corresponding pyrrolyl cases the spin density is distributed over the whole molecule, suggesting that polymerisation does not occur solely at the pyrrolyl-Cα position and the conjugation is interrupted.


Identification of somatostatin receptor type 5 gene polymorphisms associated with acromegaly.
The aim of this study was to characterize the genetic variance of somatostatin receptor 5 (SSTR5) and investigate the possible correlation of such variants with acromegaly risk and different disease characteristics. The SSTR5 gene coding region and 2000 bp upstream region was sequenced in 48 patients with acromegaly and 96 control subjects. Further, three single nucleotide polymorphisms (SNPs) were analyzed in the same group of acromegaly patients and in an additional group of 475 age- and sex-matched controls. In total, 19 SNPs were identified in the SSTR5 gene locus by direct sequencing. Three SNPs (rs34037914, rs169068, and rs642249) were significantly associated with the presence of acromegaly using the initial controls. The allele frequencies were significantly (P<0.01) different between the acromegaly patients and the additional large control group. rs34037914 and rs642249 remained significantly associated with acromegaly after Bonferroni correction and permutation tests (odds ratio (OR)=3.38; 95% confidence interval (CI), 1.78-6.42; P=0.00016 and OR=2.41; 95% CI, 1.41-4.13; P=0.0014 respectively). Haplotype reconstruction revealed two possible risk haplotypes determined by rs34037914 (633T) and rs642249 (1044A) alleles. Both haplotypes were found in significantly higher frequency in acromegaly patients compared with controls (P<0.001). In addition, the 663T allele was significantly associated with a younger age of acromegaly diagnosis (unstandardized regression coefficient β=-10.4; P=0.002), increased body mass index (β=4.1; P=0.004), higher number of adenoma resection (P<0.001) and lack of observable tumor shrinkage after somatostatin analog treatment (P=0.014). Our results demonstrate a previously undetected strong association of two SSTR5 SNPs with acromegaly. The data also suggest a possible involvement of SSTR5 variants in decreased suppression of GH production and increased tumor proliferation.


MicroRNA-92a regulates the development of cutaneous malignant melanoma by mediating FOXP1.
MicroRNAs are noncoding RNAs which are involved in the occurrence and progression of tumors. This study aims to explore the role of microRNA-92a in cutaneous malignant melanoma (CMM) and its underlying mechanism. The expression level of microRNA-92a in 75 pairs of CMM tissues and paracancerous tissues was determined using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between microRNA-92a expression with clinical data of CMM patients was analyzed. Besides, microRNA-92a expression in CMM cells and primary epidermal melanocytes (PEM) was determined by qRT-PCR as well. After transfection of si-microRNA-92a in CMM cells, biological performances of CMM were determined using cell counting kit-8 (CCK-8), colony formation and transwell assay, respectively. FOXP1 expression in CMM cells and tissues was determined using Western blot. Kaplan-Meier survival curves were drawn to explore the correlation between the FOXP1 expression and prognosis of CMM patients. MicroRNA-92a was highly expressed in CMM tissues compared with that of paracancerous tissues. Compared with CMM patients with lower expression of microRNA-92a, those with higher expression of microRNA-92a presented higher tumor stage, higher incidences of lymph node metastasis and distant metastasis, as well as lower overall survival. The knockdown of microRNA-92a remarkably decreased proliferative, invasive and metastatic capacities of CMM cells. Western blot results elucidated that microRNA-92a knockdown in CMM cells upregulates FOXP1 expression. Additionally, rescue experiments showed that mi-croRNA-92a regulates biological performances of CMM cells by regulating FOXP1. MicroRNA-92a is highly expressed in CMM, which is remarkably correlated to tumor stage and poor prognosis of CMM patients. We found that microRNA-92a pro-motes malignant progression of CMM by regulating FOXP1.


Strategies for risk-adapted therapy in myeloma.
It is clear that the clinical heterogeneity of multiple myeloma (MM) is dictated, in large part, by disease biology, predominantly genetics.(1) As novel therapeutics have emerged, and augmented our treatment armamentarium against the disease, it is increasingly important to introduce a risk-adapted approach for the optimal management of patients.(2) The selection of ideal candidates for high-dose chemotherapy with stem cell support (HDT) and maintenance will undoubtedly have to include baseline knowledge of the genetic nature of the individual. The limited duration of responses after HDT for patients with t(4;14)(p16;q32), t(14;16)(q32;q23) and 17p13 deletions highlight the need to develop a risk-adapted treatment strategy.(3)(-)(5) Novel ways of determining outcome such as the use of gene expression profiling have demonstrated differentiating capabilities not previously observed.(6) Likewise, the order of introduction of novel therapeutic agents (during induction and in the relapsing patient) will be potentially directed by similar information. As we have previously stated, MM is not only multiple but also "many."(7) Accordingly, treatment strategies will be tailored based on risk determination, genetic composition and host features.


Exonuclease proofreading by human mitochondrial DNA polymerase.
We have examined the ability of the human mitochondrial DNA polymerase to correct errors in DNA sequence using single turnover kinetic methods. The rate of excision of single-stranded DNA ranged from 0.07 to 0.17 x s(-1), depending on the identity of the 3'-base. Excision of the 3'-terminal base from correctly base paired DNA occurred at a rate of 0.05 x s(-1), indicating that the cost of proofreading is minimal, as defined by the ratio of the k(exo) for correctly base-paired DNA divided by the rate of forward polymerization (0.05/37 = 0.14%). Excision of duplex DNA containing 1-7 mismatches was biphasic, and the rate and amplitude of the fast phase increased with the number of mismatches, reaching a maximum of 9 x s(-1). We showed that transfer of DNA from the polymerase to the exonuclease active site and back again occurs through an intramolecular reaction, allowing for a complete cycle of reactions for error correction. For DNA containing a buried mismatch (T:T followed by C:G base pairs), the 3' base was removed at a rate of 3 x s(-1). The addition of nucleotide to the reaction that is identical to the 3' base increased the rate of excision 7-fold to 21 x s(-1). We propose that the free nucleotide enhances the rate of transfer of the DNA to the exonuclease active site by interrupting the correct 3' base pair through interaction with the template base. The exonuclease contribution to fidelity is minimal if the calculation is based on hydrolysis of a single mismatch: (k(exo) + k(pol,over))/(k(pol,over)) = 10, but this value increases to approximately 200 when examining error correction in the presence of nucleotides.


Delta 9-tetrahydrocannabinol and cannabinol activate capsaicin-sensitive sensory nerves via a CB1 and CB2 cannabinoid receptor-independent mechanism.
Although Delta(9)-tetrahydrocannabinol (THC) produces analgesia, its effects on nociceptive primary afferents are unknown. These neurons participate not only in pain signaling but also in the local response to tissue injury. Here, we show that THC and cannabinol induce a CB(1)/CB(2) cannabinoid receptor-independent release of calcitonin gene-related peptide from capsaicin-sensitive perivascular sensory nerves. Other psychotropic cannabinoids cannot mimic this action. The vanilloid receptor antagonist ruthenium red abolishes the responses to THC and cannabinol. However, the effect of THC on sensory nerves is intact in vanilloid receptor subtype 1 gene knock-out mice. The THC response depends on extracellular calcium but does not involve known voltage-operated calcium channels, glutamate receptors, or protein kinases A and C. These results may indicate the presence of a novel cannabinoid receptor/ion channel in the pain pathway.


Neuronal expression of Fig4 is both necessary and sufficient to prevent spongiform neurodegeneration.
FIG4 is a ubiquitously expressed phosphatase that, in complex with FAB1/PIKFYVE and VAC14, regulates the biosynthesis of the signaling lipid PI(3,5)P(2). Null mutation of Fig4 in the mouse results in spongiform degeneration of brain and peripheral ganglia, defective myelination and juvenile lethality. Partial loss-of-function of human FIG4 results in a severe form of Charcot-Marie-Tooth neuropathy. Neurons from null mice contain enlarged vacuoles derived from the endosome/lysosome pathway, and astrocytes accumulate proteins involved in autophagy. Other cellular defects include astrogliosis and microgliosis. To distinguish the contributions of neurons and glia to spongiform degeneration in the Fig4 null mouse, we expressed Fig4 under the control of the neuron-specific enolase promoter and the astrocyte-specific glial fibrillary acidic protein promoter in transgenic mice. Neuronal expression of Fig4 was sufficient to rescue cellular and neurological phenotypes including spongiform degeneration, gliosis and juvenile lethality. In contrast, expression of Fig4 in astrocytes prevented accumulation of autophagy markers and microgliosis but did not prevent spongiform degeneration or lethality. To confirm the neuronal origin of spongiform degeneration, we generated a floxed allele of Fig4 and crossed it with mice expressing the Cre recombinase from the neuron-specific synapsin promoter. Mice with conditional inactivation of Fig4 in neurons developed spongiform degeneration and the full spectrum of neurological abnormalities. The data demonstrate that expression of Fig4 in neurons is necessary and sufficient to prevent spongiform degeneration. Therapy for patients with FIG4 deficiency will therefore require correction of the deficiency in neurons.


Tumour necrosis factor (TNF-alpha) and neurological disorders in HIV infection.
Tumour necrosis factor (TNF-alpha) concentrations were determined in the CSF from 42 HIV-infected patients, with or without CNS involvement. In addition, 14 subjects with various neurological disorders but without HIV antibodies were included as controls. Raised CSF concentrations of TNF-alpha (greater than 40 ng/l) were detected both in patients with AIDS dementia complex (ADC) (6/9) and with CNS opportunistic infections (10/19) and, less commonly, in HIV infected subjects without CNS diseases (2/14) and in anti-HIV negative controls (1/14). The highest CSF concentrations of TNF-alpha (greater than 100 ng/l), however, were found in seven out of eight patients with cryptococcal meningitis. Although a role for TNF-alpha in demyelinating lesions associated with ADC has been suggested, our results indicate that a clear elevation of TNF-alpha in the CSF from HIV positive patients mostly occurs in acute inflammatory disorders, such as cryptococcal meningitis.


Optogenetic control of phosphoinositide metabolism.
Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase(OCRL)), which acts on PI(4,5)P(2) and PI(3,4,5)P(3), was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase(OCRL) to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P(2) and PI(3,4,5)P(3) biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase(OCRL) recruitment and PI(4,5)P(2) dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P(2) biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P(3) synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.


Quantum proteolysis by neutrophils: implications for pulmonary emphysema in alpha 1-antitrypsin deficiency.
Traditional enzyme kinetics provide a poor explanation for the increased risk of lung injury in alpha 1-antitrypsin (AAT) deficiency. Millimolar concentrations of leukocyte elastase, when released from single azurophil granules of activated neutrophils, lead to evanescent quantum bursts of proteolytic activity before catalysis is quenched by pericellular inhibitors. Herein, we tested the possibility that quantum proteolytic events are abnormal in AAT deficiency. We incubated neutrophils on opsonized fluoresceinated fibronectin in serum from individuals with various AAT phenotypes, and then measured and modeled quantum proteolytic events. The mean areas of the events in serum from heterozygous individuals (Pi MZ and Pi SZ) were slightly, but significantly, larger than those in serum from normal patients (Pi M). In marked contrast, mean areas of events in serum from AAT-deficient individuals were 10-fold larger than those in serum from normal patients. Diffusion modeling predicted that local elastase concentrations exceed AAT concentrations for less than 20 milliseconds and for more than 80 milliseconds in Pi M and Pi Z individuals, respectively. Thus, quantum proteolytic events are abnormally large and prolonged in AAT deficiency, leading directly to an increased risk of tissue injury in the immediate vicinity of activated neutrophils. These results have potentially important implications for the pathogenesis and prevention of lung disease in AAT deficiency.


Examination of spatial polygamy among young gay, bisexual, and other men who have sex with men in New York City: the P18 cohort study.
The few previous studies examining the influence of the neighborhood context on health and health behavior among young gay, bisexual, and other men who have sex with men (YMSM) have predominantly focused on residential neighborhoods. No studies have examined multiple neighborhood contexts among YMSM or the relationships between sociodemographic characteristics, psychosocial factors, social support network characteristics, health behaviors, and neighborhood concordance. In this study, we assessed spatial polygamy by determining the amount of concordance between residential, social, and sex neighborhoods (defined as boroughs) in addition to examining individual-level characteristics that may be associated with neighborhood concordance. These data come from the baseline assessment of Project 18, a cohort of racially and ethnically diverse YMSM residing in the New York City metropolitan area. Participants (N = 598) provided information on their residential, social, and sex boroughs as well as information on their sociodemographic characteristics, psychosocial factors, social support network characteristics, and health behaviors (e.g., substance use and condomless sex). Descriptive analyses were conducted to examine the distribution of boroughs reported across all three contexts, i.e., residential, social, and sex boroughs. Next, concordance between: (1) residential and social boroughs; (2) residential and sex boroughs; (3) social and sex boroughs; and (4) residential, social, and sex boroughs was assessed. Finally, bivariable analyses were conducted to examine the relationships between sociodemographic characteristics, psychosocial factors, social support network characteristics, and health behaviors in relation to borough concordance. Approximately two-thirds of participants reported concordance between residential/socializing, residential/sex, and sex/socializing boroughs, whereas 25% reported concordance between all three residential/socializing/sex boroughs. Borough concordance varied by some individual-level characteristics. For example, White YMSM and YMSM reporting lower perceived socioeconomic status were significantly more likely to report residential/socializing/sex borough concordance (p < 0.001). With regard to psychosocial factors, YMSM who reported experiencing gay-related stigma in public forums were more likely to report discordant socializing/sex and residential/socializing/sex boroughs (p < 0.001). Greater frequency of communication with network members (>weekly) was associated with less residential/social borough concordance (p < 0.05). YMSM who reported residential/socializing/sex borough concordance were more likely to report recent (last 30 days) alcohol use, recent marijuana use, and recently engaging in condomless oral sex (all p < 0.05). These findings suggest that spatial polygamy, or an individual moving across and experiencing multiple neighborhood contexts, is prevalent among urban YMSM and that spatial polygamy varies by multiple individual-level characteristics. Future research among YMSM populations should consider multiple neighborhood contexts in order to provide a more nuanced understanding of how and which neighborhood contexts influence the health and well-being of YMSM. This further examination of spatial polygamy (and individual-level characteristics associated with it) may increase understanding of the most appropriate locations for targeted disease prevention and health promotion interventions (e.g., HIV prevention interventions).


Influence of horizontally curved roadway section characteristics on motorcycle-to-barrier crash frequency.
The purpose of this study was to investigate motorcycle-to-barrier crash frequency on horizontally curved roadway sections in Washington State using police-reported crash data linked with roadway data and augmented with barrier presence information. Data included 4915 horizontal curved roadway sections with 252 of these sections experiencing 329 motorcycle-to-barrier crashes between 2002 and 2011. Negative binomial regression was used to predict motorcycle-to-barrier crash frequency using horizontal curvature and other roadway characteristics. Based on the model results, the strongest predictor of crash frequency was found to be curve radius. This supports a motorcycle-to-barrier crash countermeasure placement criterion based, at the very least, on horizontal curve radius. With respect to the existing horizontal curve criterion of 820 feet or less, curves meeting this criterion were found to increase motorcycle-to-barrier crash frequency rate by a factor of 10 compared to curves not meeting this criterion. Other statistically significant predictors were curve length, traffic volume and the location of adjacent curves. Assuming curves of identical radius, the model results suggest that longer curves, those with higher traffic volume, and those that have no adjacent curved sections within 300 feet of either curve end would likely be better candidates for a motorcycle-to-barrier crash countermeasure.