Phosphorus in agricultural soils: drivers of its distribution at the global scale.
Phosphorus (P) availability in soils limits crop yields in many regions of the World, while excess of soil P triggers aquatic eutrophication in other regions. Numerous processes drive the global spatial distribution of P in agricultural soils, but their relative roles remain unclear. Here, we combined several global data sets describing these drivers with a soil P dynamics model to simulate the distribution of P in agricultural soils and to assess the contributions of the different drivers at the global scale. We analysed both the labile inorganic P (PILAB ), a proxy of the pool involved in plant nutrition and the total soil P (PTOT ). We found that the soil biogeochemical background corresponding to P inherited from natural soils at the conversion to agriculture (BIOG) and farming practices (FARM) were the main drivers of the spatial variability in cropland soil P content but that their contribution varied between PTOT vs. PILAB . When the spatial variability was computed between grid cells at half-degree resolution, we found that almost all of the PTOT spatial variability could be explained by BIOG, while BIOG and FARM explained 38% and 63% of PILAB spatial variability, respectively. Our work also showed that the driver contribution was sensitive to the spatial scale characterizing the variability (grid cell vs. continent) and to the region of interest (global vs. tropics for instance). In particular, the heterogeneity of farming practices between continents was large enough to make FARM contribute to the variability in PTOT at that scale. We thus demonstrated how the different drivers were combined to explain the global distribution of agricultural soil P. Our study is also a promising approach to investigate the potential effect of P as a limiting factor for agroecosystems at the global scale.


Progress in evidence-based medicine: a quarter century on.
In response to limitations in the understanding and use of published evidence, evidence-based medicine (EBM) began as a movement in the early 1990s. EBM's initial focus was on educating clinicians in the understanding and use of published literature to optimise clinical care, including the science of systematic reviews. EBM progressed to recognise limitations of evidence alone, and has increasingly stressed the need to combine critical appraisal of the evidence with patient's values and preferences through shared decision making. In another progress, EBM incorporated and further developed the science of producing trustworthy clinical practice guidelines pioneered by investigators in the 1980s. EBM's enduring contributions to clinical medicine include placing the practice of medicine on a solid scientific basis, the development of more sophisticated hierarchies of evidence, the recognition of the crucial role of patient values and preferences in clinical decision making, and the development of the methodology for generating trustworthy recommendations.


Usefullness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.
In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society.


Medication reconciliation at admission and discharge: a time and motion study.
Medication reconciliation at admission, transfer and discharge has been designated as a required hospital practice to reduce adverse drug events. However, implementation challenges have resulted in poor hospital adherence. The aim of this study was to assess the processes required to carry out medication reconciliation: the health professionals involved, the tasks and time devoted to medication reconciliation in general hospital settings. A time-and-motion study design was used. Using a systematic sample of patients admitted and discharged from geriatric, medical and surgical units in two academic centers, health professionals involved in medication reconciliation were observed and timed. Descriptive statistics were used to summarize the number of professionals involved, tasks performed, and mean time devoted. Up to 3 professionals from 2 disciplines (medicine and pharmacy) were involved in the medication reconciliation process. Geriatric reconciliations took the most time to complete at admission (mean: 92.2 minutes (SD = 44.3)) and discharge (mean: 29.0 minutes (SD = 23.8)), followed by internal medicine at admission (mean: 46.2 minutes (SD = 21.1)) and 19.4 (SD = 11.7) minutes at discharge) and general surgery minutes at discharge (mean: 9.9 minutes (SD = 18.2)). Considerable differences in order, type and number of tasks performed were noted between and within units. Tasks independent of direct patient interaction took more than twice the time required to complete than tasks requiring patient interaction. Lack of coordination, specialized training and agreement on the roles and responsibilities of professionals are among the most probable reasons for work-flow inefficiencies, possibly variability in quality, and time required for the current medication reconciliation process. A better understanding of the admission processes in general surgery is required. Standardization and use of electronic tools could improve efficiency and hospital adherence.


Uncorrelated healing response of tendon and ear injuries in MRL highlight a role for the local tendon environment in driving scarless healing.
Tendon tears are common injuries that heal with scar formation. Interestingly, MRL/MpJ mice heal without scar in several tissues, including tendon. Most hypotheses regarding scarless healing implicate the systemic environment. However, the tissue-specificity of this regenerative response and our previous findings showing regeneration of sub-rupture tendon injuries, which lack an overt systemic response, motivate a tissue-driven hypothesis. Our objective is to investigate the potential of the local tendon environment in driving scarless healing (1) by comparing the systemic response and the healing capacity associated with ear and tendon injuries in MRL/MpJ mice, and (2) by comparing intrinsic healing properties between MRL/MpJ and normal healer C57Bl/6 tendons. We examined the systemic inflammatory and local structural environments of ear and tendon punch injuries in MRL/MpJ and C57Bl/6 mice. Systemic differences were analyzed to assess effects of different injuries on the inflammatory response. Correlations were assessed between MRL/MpJ ear and tendon injuries to compare the extent of healing between regenerative tissues. Analysis showed similarities between the systemic environment in MRL/MpJ post ear or tendon injuries. However, comparable inflammatory responses did not translate into analogous healing between tissues, suggesting that the systemic environment is not the driver of regeneration. Supporting the regenerative role of the local environment, healing MRL/MpJ tendons exhibited improved matrix and cell alignment and a distinct composition of growth factors and Hyaluronan from C57Bl/6. These findings support the tissue-driven hypothesis for MRL/MpJ tendon regeneration and motivate further investigation regarding specific roles of extracellular factors in scarless healing.


The relationship between the emergent primary percutaneous coronary intervention quality measure and inpatient myocardial infarction mortality.
In the setting of acute ST-segment elevation myocardial infarction (STEMI), reperfusion therapy with emergent primary percutaneous coronary intervention (PCI) significantly reduces mortality. It is unknown whether a hospital's performance on the Centers for Medicare & Medicaid Services (CMS) quality metric for time from patient arrival to angioplasty is associated with its overall hospital acute myocardial infarction (AMI) mortality rate. The objective of this study was to evaluate if hospitals with higher performance on the time-to-PCI quality measure are more likely to achieve lower mortality for patients admitted for any type of AMI. Using merged 2006 data from the Nationwide Inpatient Sample (NIS), the American Hospital Association (AHA) annual survey, and CMS Hospital Compare quality indicator data, we examined 69,101 admissions with an International Classification of Diseases, Ninth Revision (ICD-9)-coded principal diagnosis of AMI in the 116 hospitals that reported more than 24 emergent primary PCI admissions in that year. Hospitals were categorized into quartiles according to percentage of admissions in 2006 that achieved the primary PCI timeliness threshold (time-to-PCI quality measure). Using a random effects logistic regression model of inpatient mortality, we examined the significance of the hospital time-to-PCI quality measure after adjustment for other hospital and individual patient sociodemographic and clinical characteristics. The unadjusted inpatient AMI mortality rate at the 27 top quartile hospitals was 4.3%, compared to 5.1% at the 32 bottom quartile (worst performing) hospitals. The risk-adjusted odds ratio (OR) of inpatient death was 0.83 (95% confidence interval [CI] = 0.72 to 0.95), or 17% lower odds of inpatient death, among patients admitted to hospitals in the top quartile for the time-to-PCI quality measure compared to the case if the hospitals were in the bottom 25th percentile. Hospitals with the highest and second highest quartiles of time-to-PCI quality measure had a significantly lower overall AMI mortality rate than the lowest quartile hospitals. Despite the fact that a minority of all patients with AMI get an emergent primary PCI, hospitals that perform this more efficiently also had a significantly lower mortality rate for all their patients admitted with AMI. The time-to-PCI quality measure in 2006 was a potentially important proxy measure for overall AMI quality of care.


Activation of KCNQ Channels Suppresses Spontaneous Activity in Dorsal Root Ganglion Neurons and Reduces Chronic Pain after Spinal Cord Injury.
A majority of people who have sustained spinal cord injury (SCI) experience chronic pain after injury, and this pain is highly resistant to available treatments. Contusive SCI in rats at T10 results in hyperexcitability of primary sensory neurons, which contributes to chronic pain. KCNQ channels are widely expressed in nociceptive dorsal root ganglion (DRG) neurons, are important for controlling their excitability, and their activation has proven effective in reducing pain in peripheral nerve injury and inflammation models. The possibility that activators of KCNQ channels could be useful for treating SCI-induced chronic pain is strongly supported by the following findings. First, SCI, unlike peripheral nerve injury, failed to decrease the functional or biochemical expression of KCNQ channels in DRG as revealed by electrophysiology, real-time quantitative polymerase chain reaction, and Western blot; therefore, these channels remain available for pharmacological targeting of SCI pain. Second, treatment with retigabine, a specific KCNQ channel opener, profoundly decreased spontaneous activity in primary sensory neurons of SCI animals both in vitro and in vivo without changing the peripheral mechanical threshold. Third, retigabine reversed SCI-induced reflex hypersensitivity, adding to our previous demonstration that retigabine supports the conditioning of place preference after SCI (an operant measure of spontaneous pain). In contrast to SCI animals, naïve animals showed no effects of retigabine on reflex sensitivity or conditioned place preference by pairing with retigabine, indicating that a dose that blocks chronic pain-related behavior has no effect on normal pain sensitivity or motivational state. These results encourage the further exploration of U.S. Food and Drug Administration-approved KCNQ activators for treating SCI pain, as well as efforts to develop a new generation of KCNQ activators that lack central side effects.


The opsonin MFG-E8 is a ligand for the alphavbeta5 integrin and triggers DOCK180-dependent Rac1 activation for the phagocytosis of apoptotic cells.
Opsonization of apoptotic cells facilitates recognition by phagocytes for the rapid clearance of potentially inflammatory cellular material. The secreted glycoprotein Milk Fat Globule Factor-E8 (MFG-E8) is a member of this family of bridging molecules and is believed to bind phosphatidylserine (PS) on the dying cell, linking it to integrin receptors on the phagocyte. Here we report the characterization of a functional signaling module involving MFG-E8, alphavbeta5 integrin, and DOCK180 for the activation of Rac1. We show that MFG-E8 and DOCK180 are both expressed in phagocytic-competent primary immature dendritic cells (DCs) and DC2.4 cells, and are potently down-regulated upon DC maturation, consistent with their role in phagocytosis and antigen capture. Coexpression of MFG-E8 with alphavbeta5 integrin potentiated integrin-mediated Rac1 activation, which was abrogated by mutagenesis in the RGD motif in MFG-E8. Moreover, expression of antisense DOCK180 abrogated MFG-E8-alphavbeta5-mediated Rac activation and impaired the phagocytosis of apoptotic cells. These data demonstrate a biochemical link between an opsonin of apoptotic cells, the alphavbeta5 integrin, and the Crk-DOCK180-Rac1 pathway, and importantly, show that MFG-E8 and DOCK180 are expressed according to the functional status of the phagocyte.


Effects of acupuncture at ST36 on pharmacokinetics of Schisandra lignans in rats.
To investigate the influence of acupuncture at ST36 on the pharmacokinetics of Schisandra lignans including schisandrin, deoxyschisandrin and schisandrin B after intragastric administration of Schisandra chinensis (SC) in rats. Twelve male Sprague-Dawley rats were randomly divided into two study groups: SC and SC+acupuncture. Rats in both groups received intragastric SC extract at 5.0 g/kg. Rats in the SC+acupuncture group additionally received acupuncture stimulation at ST36 for 30 min after SC administration. Acupuncture needles were rotated bilaterally for 1 min, left in situ for 20 min, then electrically stimulated for 10 min at 50 Hz frequency and 1-3 mA intensity. A sensitive and specific high performance liquid chromatography electrospray tandem mass spectrometry procedure was developed and validated for simultaneous analysis of three bioactive lignans (schisandrin, deoxyschisandrin and schisandrin B) in rat plasma. There were significant differences (p<0.05) between the two study groups in various pharmacokinetic parameters. Area under the plasma concentration-time curve (AUC0-t), area under the plasma concentration-time curve to time infinity (AUC0-∞) and peak plasma concentration (Cmax) for schisandrin, absorption half-life (T1/2α) and AUC0-t for deoxyschisandrin, and Cmax for schisandrin B were increased in the SC+acupuncture group compared with the SC group. T1/2α for schisandrin B only and time to peak concentration (Tmax) for all three lignans were reduced following acupuncture. Acupuncture stimulation at ST36 affects the pharmacokinetics of SC in rats. Acupuncture may have a beneficial role in promoting the absorption of lignans from extracts of SC.


Faces in a crowd: high socially anxious individuals estimate that more people are looking at them than low socially anxious individuals.
People with social anxiety disorder are afraid of being scrutinized by others and often feel that they are the excessive focus of other people's attention. This study investigated whether, when compared to low socially anxious individuals, high socially anxious individuals overestimate the proportion of people in a crowd who are observing them. It was hypothesized that any potential overestimation would be modulated by self-focused attention. Forty-eight high and 48 low socially anxious participants performed a "faces in a crowd" computer task during which they briefly saw matrices of faces, which varied in terms of the proportion of people who were looking at them. Participants estimated the proportion of people who were looking at them. The task was performed once with mirrors present (to induce an enhanced self-focused state) and once without mirrors present (neutral state). Participants' subjective estimates and the objective proportion of faces looking towards them were strongly correlated in both the high and low socially anxious groups. However, high socially anxious participants estimated that more people were looking at them than low socially anxious participants. In the first phase of the experiment, but not in the later phases, this effect was magnified in the mirror condition. This study provides preliminary evidence of a social anxiety related perceptual difference that may be amplified by self-focused attention. Clinical implications are discussed.


CD1a selectively captures endogenous cellular lipids that broadly block T cell response.
We optimized lipidomics methods to broadly detect endogenous lipids bound to cellular CD1a proteins. Whereas membrane phospholipids dominate in cells, CD1a preferentially captured sphingolipids, especially a C42, doubly unsaturated sphingomyelin (42:2 SM). The natural 42:2 SM but not the more common 34:1 SM blocked CD1a tetramer binding to T cells in all human subjects tested. Thus, cellular CD1a selectively captures a particular endogenous lipid that broadly blocks its binding to TCRs. Crystal structures show that the short cellular SMs stabilized a triad of surface residues to remain flush with CD1a, but the longer lipids forced the phosphocholine group to ride above the display platform to hinder TCR approach. Whereas nearly all models emphasize antigen-mediated T cell activation, we propose that the CD1a system has intrinsic autoreactivity and is negatively regulated by natural endogenous inhibitors selectively bound in its cleft. Further, the detailed chemical structures of natural blockers could guide future design of therapeutic blockers of CD1a response.


Hyperlipidaemia in HIV-infected patients on lopinavir/ritonavir monotherapy in resource-limited settings.
Cardiovascular disease (CVD) is an emerging concern for HIV-infected patients. Hyperlipidaemia is a risk factor for CVD and a complication of protease-inhibitor-based antiretroviral therapy, but little is known about its incidence and risk factors in treated patients in resource-limited settings (RLS). We conducted a secondary analysis of ACTG A5230 trial in which HIV-infected adults from India, Malawi, Tanzania, Thailand and South Africa, with virological relapse on first-line therapy were initiated on lopinavir/ritonavir (LPV/r) monotherapy. Hyperlipidaemia was a grade 2+ elevated fasting total cholesterol (FTC≥240 mg/dl) or fasting triglycerides (FTG≥500 mg/dl) or calculated low-density lipoprotein cholesterol (LDL≥160 mg/dl) based on measurements at weeks 12, 24, 48, 68 and 104. We evaluated factors potentially associated with quantitative lipid changes from baseline to week 12. These were age, sex, race, site and baseline body mass index, CD4+ T-cell count, HIV-1 RNA level and lipids. 106 participants without hyperlipidaemia at baseline started LPV/r; median age 39 years, 68% Black African, 55% female. The cumulative incidence of hyperlipidaemia at week 104 was 48% (95% CI 36, 58%). At week 12, there were significant mean increases from baseline in FTC (17 mg/dl, P<0.001) and FTG (104 mg/dl, P<0.001). In multivariable analysis, higher baseline FTC (P=0.044), FTG (P=0.025), Thai (P<0.001) or Indian sites (P=0.020) versus African sites were associated with increased risk of hyperlipidaemia. In HIV-infected adults in RLS initiating LPV/r, hyperlipidaemia was common. Baseline lipid measurements and routine monitoring should be recommended in individuals starting LPV/r-based treatments with borderline high lipids.


Mental, social, and physical well-being in New Hampshire, Oregon, and Washington, 2010 Behavioral Risk Factor Surveillance System: implications for public health research and practice related to Healthy People 2020 foundation health measures on well-being.
Well-being is now accepted as one of four cross-cutting measures in gauging progress for Healthy People 2020. This shift to population indicators of well-being redresses notions of health that have focused on absence of illness (negative health) as a primary or sufficient indicator of positive functioning. The purpose of this study was to estimate mental, social, and physical well-being in three US states using new measures piloted on the 2010 Behavioral Risk Factor Surveillance Survey System (BRFSS). Baseline estimates were provided for states overall, and within states for demographic subgroups, those with chronic health conditions or disabilities, and those with behavioral risk factors. Ten validated questions designed to assess mental (e.g., satisfaction with life, satisfaction with life domains, happiness), physical (e.g., satisfaction with energy level), and social dimensions (e.g., frequency of social support) of well-being were selected with state input for inclusion on BRFSS. 18,622 individuals responded to the BRFSS surveys administered by New Hampshire (N = 3,139), Oregon (N = 2,289), and Washington (N = 13,194). Multivariate adjusted proportions of positive responses to well-being items were examined. After adjustment for confounders, about 67% of adults in these states had high levels of well-being, including >80% reporting experiencing happiness. Most adults were satisfied with their work, neighborhood, and education, but significant differences were seen in subgroups. Well-being differed by demographic characteristics such as marital status, health behaviors, chronic conditions, and disability status, with those who reported a disability and smokers consistently experiencing the worst well-being. Well-being is accepted as one of four cross-cutting measures in gauging progress for Healthy People 2020. Well-being differs by important sociodemographic factors and health conditions (e.g., age, employment, smoking, disability status). These findings provide baseline estimates for the three states to use in gauging improvements in well-being and can serve as a model for other state-level or national surveillance systems. These findings also assist states in identifying vulnerable subgroups who may benefit from potential interventions such as those in the National Prevention Strategy that focus on enhancing well-being where such disparities exist.


Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions.
Fluorescence in situ hybridization (FISH) was performed on sections of plastic-embedded tissue using 16S rRNA-directed oligonucleotide probes to visualize uncultured treponemes in skin biopsies of cows with digital dermatitis. Plastic as embedding material allowed sectioning of hard and soft tissue with a defined thickness, avoiding the risk of dragging bacteria into the tissue while sectioning. furthermore, it provided a good signal-to-noise ratio. Using this method the spatial distribution of three different bacterial phylotypes was visualized simultaneously within the tissue. Whereas debris covering the ulcers contained a mixture of different micro-organisms, a layering of certain treponemal phylotypes was observed deeper in the epidermis. Confocal laser scanning microscopy and subsequent three-dimensional reconstruction of series of optical sections confirmed that the treponemes migrated intercellularly around the cells, most of them directed towards the dermis. In situ hybridization on tissue embedded in plastic proved to be a useful method to study mixed bacterial infections since it combines excellent histological conservation of tissue with identification of bacterial species by simultaneous use of probes labelled with different fluorescent dyes. This technique may have implications for in situ detection, identification and localization of microorganisms in veterinary as well as in human medicine.


Physicochemical interactions with (-)-epigallocatechin-3-gallate drive structural modification of celiac-associated peptide α2-gliadin (57-89) at physiological conditions.
(-)-Epigallocatechin-3-gallate (EGCG), a major phenolic constituent of tea, has been shown to have biological activity within inflammatory pathways involved with food allergies and intolerances. Proposed mechanisms for this effect include sequestration and structural modification of immunostimulatory proteins as a result of interactions with EGCG. The present study employs biophysical techniques including dynamic light scattering, circular dichroism and nuclear magnetic resonance to elucidate the likely mechanism(s) by which EGCG interacts with α2-gliadin (57-89) (α2g), an immunodominant peptide in celiac disease pathogenesis. We demonstrate that EGCG interacts with α2g in a multi-phase reaction driven by non-specific binding, resulting in the formation of polydisperse EGCG/α2g complexes which induce changes in peptide structure. We also show that these interactions occur at a range of pH levels associated with digestion, including pH 2.0, 6.8 and 7.5. Based on previous reports of binding specificity of enzymes and antigen presenting cells in celiac disease pathogenesis, our results provide foundational support for EGCG to prevent recognition of immunostimulatory gliadin epitopes by the body and thus prevent the inflammatory and autoimmune response associated with celiac disease.


Effect of resin-modified glass ionomer cements on secondary caries.
To evaluate the in vitro secondary caries inhibitory effect of two resin-modified glass ionomer cements (R-GICs). Class V cavities were prepared at the cementoenamel junction on facial and lingual surfaces of 32 extracted upper premolars. The facial cavities were restored with a conventional glass ionomer cement (GIC) (Fuji II), while the lingual cavities were restored with either one of the R-GICs (Fuji II LC, Photac Fil, Vitremer), or a resin composite (Z-100). The margins of the restorations were subjected to a 20-day in vitro cariogenic challenge and the degree of demineralization was analyzed by microradiography. The amounts of fluoride released into distilled water from disc specimens of the materials tested were also measured for immersion time up to 161 days. The R-GICs and GIC's released similar cumulative amounts of fluoride over 161 days. Photac-Fil showed significantly higher amounts of fluoride release over the same period. The depth of the outer lesion and the thickness of the acid-resistant layer showed no significant difference among the R-GICs and GIC's. Moreover, the residual fluoride and calcium in the dentin adjacent to the R-GICs and GIC's were correlated with the thickness of acid-resistant layers in the dentin adjacent to the R-GICs and GIC's by electron probe microanalysis. However, the inhibitory effect of the R-GICs was not directly related to the fluoride concentrations eluted from them.


Cyclooxygenase-2-mediated DNA damage.
Rat intestinal epithelial cells that express the cyclooxygenase-2 (COX-2) gene permanently (RIES cells) were used as a model of in vivo oxidative stress. A targeted lipidomics approach showed that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) was the major hydroxylated non-esterified lipid formed in unstimulated intact cells. The corresponding hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15(S)-HPETE) undergoes homolytic decomposition to the DNA-reactive bifunctional electrophile 4-oxo-2(E)-nonenal, a precursor of heptanone-etheno-2'-deoxyguanosine. This etheno adduct was identified in the DNA of RIES cells. A dose-dependent increase in adduct levels was observed in the presence of vitamin C. This suggested that vitamin C increased lipid hydroperoxide-mediated 4-oxo-2(E)-nonenal formation in the cells. The selective COX-2 inhibitor NS-398 was protective against cellular DNA damage but was less effective if vitamin C was present. Prostaglandin E(2) and 15(S)-HETE biosynthesis were completely inhibited by 110 mum NS-398 in the intact RIES cells. No inhibition of COX-1 was detected in the wild-type RIE cells at this concentration of NS-398. Arachidonic acid treatment of RIES cell lysates and ionophore stimulation of intact RIES cells produced significantly more 15(R)-HETE than the untreated intact cells. These preparations also both produced 11(R)-HETE, which was not detected in the intact cells. Aspirin treatment of the intact unstimulated RIES cells resulted in the exclusive formation of 15(R)-HETE in amounts that were slightly higher than the original 15(S)-HETE observed in the absence of aspirin, implying that significant amounts of 15(R)-HPETE had also been formed. 15(R)-HPETE should give exactly the same amount of heptanone-etheno-2'-deoxyguanosine as its 15(S)-enantiomer. However, no increase in heptanone-etheno adduct formation occurred in the aspirin-treated cells. The present study suggests a potential mechanism of tumorigenesis that involves DNA adduct formation from COX-2-mediated lipid peroxidation rather than prostaglandin formation. Therefore, inhibition of COX-2-mediated lipid hydroperoxide formation offers a potential therapeutic alternative to COX-2 inhibitors in chemoprevention strategies.


Antenatal dexamethasone treatment transiently alters diastolic function in the mouse fetal heart.
Endogenous glucocorticoid action is important in the structural and functional maturation of the fetal heart. In fetal mice, although glucocorticoid concentrations are extremely low before E14.5, glucocorticoid receptor (GR) is expressed in the heart from E10.5. To investigate whether activation of cardiac GR prior to E14.5 induces precocious fetal heart maturation, we administered dexamethasone in the drinking water of pregnant dams from E12.5 to E15.5. To test the direct effects of glucocorticoids upon the cardiovascular system we used SMGRKO mice, with Sm22-Cre-mediated disruption of GR in cardiomyocytes and vascular smooth muscle. Contrary to expectations, echocardiography showed no advancement of functional maturation of the fetal heart. Moreover, litter size was decreased 2 days following cessation of antenatal glucocorticoid exposure, irrespective of fetal genotype. The myocardial performance index and E/A wave ratio, markers of fetal heart maturation, were not significantly affected by dexamethasone treatment in either genotype. Dexamethasone treatment transiently decreased the myocardial deceleration index (MDI; a marker of diastolic function), in control fetuses at E15.5, with recovery by E17.5, 2 days after cessation of treatment. MDI was lower in SMGRKO than in control fetuses and was unaffected by dexamethasone. The transient decrease in MDI was associated with repression of cardiac GR in control fetuses following dexamethasone treatment. Measurement of glucocorticoid levels in fetal tissue and hypothalamic corticotropin-releasing hormone (Crh) mRNA levels suggest complex and differential effects of dexamethasone treatment upon the hypothalamic-pituitary-adrenal axis between genotypes. These data suggest potentially detrimental and direct effects of antenatal glucocorticoid treatment upon fetal heart function.


Prolonged Antiretroviral Treatment Induces Adipose Tissue Remodelling Associated with Mild Inflammation in SIV-Infected Macaques.
During chronic SIV/HIV infection, adipose tissue (AT) is the target of both antiretroviral treatment (ART) and the virus. AT might subsequently contribute to the low-grade systemic inflammation observed in patients on ART. To evaluate the inflammatory profile of AT during chronic SIV/HIV infection, we assayed subcutaneous and visceral abdominal AT from non-infected (SIV-, control), ART-naïve SIV-infected (SIV+) and ART-controlled SIV-infected (SIV+ART+) cynomolgus macaques for the mRNA expression of genes coding for factors related to inflammation. Significant differences were observed only when comparing the SIV+ART+ group with the SIV+ and/or SIV- groups. ART-treated infection impacted the metabolic fraction (with elevated expression of PPARγ and CEBPα), the extracellular matrix (with elevated expression of COL1A2 and HIF-1α), and the inflammatory profile. Both pro- and anti-inflammatory signatures were detected in AT, with greater mRNA expression of anti-inflammatory markers (adiponectin and CD163) and markers associated with inflammation (TNF-α, Mx1, CCL5 and CX3CL1). There were no intergroup differences in other markers (IL-6 and MCP-1). In conclusion, we observed marked differences in the immune and metabolic profiles of AT in the context of an ART-treated, chronic SIV infection; these differences were related more to ART than to SIV infection per se.


Endogenous adenosine restrains renin release in conscious rats.
The purpose of this study was to test the hypothesis that endogenous adenosine functions to restrain the renin release response to pharmacological and pathophysiological stimuli. To achieve this objective, we examined the effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl)xanthine (DPSPX), on the renin release response induced by acute administration of hydralazine or by chronic clipping of the left renal artery (renovascular hypertensive rats). In conscious, unrestrained rats, DPSPX significantly increased plasma renin activity (PRA) in control rats, in rats treated with hydralazine, and in renovascular hypertensive rats. The effect of DPSPX on PRA was significantly greater in rats treated with hydralazine or in renovascular hypertensive rats compared with control rats. DPSPX did not influence arterial blood pressure in any group, did not affect the measurement of PRA, and did not alter the elimination of renin activity from the circulation. Additional experiments were performed in the in situ autoperfused kidney so that the effects of DPSPX on renal hemodynamics and renal excretory function could be assessed. In this experimental model, DPSPX also increased PRA in hydralazine-treated rats and in renovascular hypertensive rats without affecting arterial pressure, renal blood flow, or sodium excretion. In a final set of studies in conscious, unrestrained rats, adenosine deaminase increased PRA in a dose-dependent manner in hydralazine-treated rats and significantly increased the slope of the relation between PRA and the depressor response to hydralazine. We conclude: 1) Although the kidney has both A1 and A2 adenosine receptors mediating inhibitory and stimulatory actions, respectively, on renin release, the dominant effect of endogenous adenosine on renin release is inhibitory. 2) Even under basal physiological conditions, endogenous adenosine tonically inhibits renin release. 3) This inhibitory effect is augmented whenever the renin-angiotensin system is stimulated regardless of the approach used to activate renin release. 4) Endogenous adenosine negatively modulates renin release by a direct effect on juxtaglomerular cells.


FOLLOWING AND IMPRINTING: EFFECTS OF LIGHT AND SOCIAL EXPERIENCE.
Ninety-six Vantress broiler chicks were used in two studies of the effects of light and social experience on the "following" response. In addition, the animals used in one study were later tested to determine the amount of imprinting during following. The difference between the behavior of these animals and animals maintained in isolation and in darkness prior to exposure to a model, indicate that variability in treatment can influence the following behavior, and that following in itself cannot be equated with imprinting.


The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis.
The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells. In this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis thaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1 recombinase successfully excised the target sequence in a conservative manner and the resulting recombination event was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In addition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target transgenic plants to generate excised progeny. The Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana germinal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1 in planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without selectable marker transgenes or other undesirable exogenous sequences.


Two New Species of Liuixalus (Rhacophoridae, Anura): Evidence from Morphological and Molecular Analyses.
Due to small body sizes, superficial similarities in morphologies, and obscure activity behaviors, the phylogeny and taxonomy of species in the genus Liuixalus were very troublesome. Some species might comprise a complex of cryptic species. To investigate the species of group, we constructed the matrilineal genealogy of the genus using 16s rRNA mitochondrial DNA sequences. Analyses recovered six well supported matrilines that involved L. romeri, L. ocellatus, L. hainanus, L. calcarius, Liuixalus shiwandashan sp. nov. and Liuixalus jinxiuensis sp. nov., though the historical relationships among them remained unresolved. Currently, Liuixalus included 4 species, distributed eastwards from northern Vietnam to Hong Kong, China. Based on genealogical and morphological distinctiveness, we described Liuixalus jinxiuensis sp. nov. from the type locality Mt. Dayao, Jinxiu, China and Liuixalus shiwandashan sp. nov. from the type locality Mt. Shiwanda, China. A combination of morphological measurements, genetic, bioacoustic and osteological analysis was served to diagnose the new taxa.


Disks around merging binary black holes: From GW150914 to supermassive black holes.
We perform magnetohydrodynamic simulations in full general relativity of disk accretion onto nonspinning black hole binaries with mass ratio q = 29/36. We survey different disk models which differ in their scale height, total size and magnetic field to quantify the robustness of previous simulations on the initial disk model. Scaling our simulations to LIGO GW150914 we find that such systems could explain possible gravitational wave and electromagnetic counterparts such as the Fermi GBM hard x-ray signal reported 0.4 s after GW150915 ended. Scaling our simulations to supermassive binary black holes, we find that observable flow properties such as accretion rate periodicities, the emergence of jets throughout inspiral, merger and postmerger, disk temperatures, thermal frequencies, and the time delay between merger and the boost in jet outflows that we reported in earlier studies display only modest dependence on the initial disk model we consider here.


[Can we prescribe lithium during pregnancy? Summary of a controversy].
Lithium is the standard treatment for bipolar disorder. It has long been contra-indicated during pregnancy because of its teratogenicity, especially regarding cardiac anomalies. More recent epidemiological studies show that the teratogenic risk of lithium has been overestimated in the past. When caring for a pregnant bipolar woman, physicians should consider the risk of fetal exposure to a mood stabilizer but also the risk of relapse if the treatment is interrupted. We have conducted a literature review on this subject. In the second part of the article, we have looked at the authors' recommendations about possible administration of lithium during pregnancy, timing of prophylaxis and precautions to minimize risks.


The angiogenic makeup of human hepatocellular carcinoma does not favor vascular endothelial growth factor/angiopoietin-driven sprouting neovascularization.
Quantitative data on the expression of multiple factors that control angiogenesis in hepatocellular carcinoma (HCC) are limited. A better understanding of the mechanisms underlying angiogenesis in HCC will improve the rational choice of anti-angiogenic treatment. We quantified gene and protein expression of members of the vascular endothelial growth factor (VEGF) and angiopoietin systems and studied localization of VEGF, its receptors VEGFR-1 and VEGFR-2, Angiopoietin (Ang)-1 and Ang-2, and their receptor, in HCC in noncirrhotic and cirrhotic livers. We employed real-time reverse transcription polymerase chain reaction (RT-PCR), western blot, and immunohistology, and compared the outcome with highly angiogenic human renal cell carcinoma (RCC). HCC in noncirrhotic and cirrhotic livers expressed VEGF and its receptors to a similar extent as normal liver, although in cirrhotic background, VEGFR-2 levels in both tumor and adjacent tissue were decreased. Ang-1 expression was slightly increased compared with normal liver, whereas Tie-2 was strongly down-regulated in the tumor vasculature. Ang-2 messenger RNA (mRNA) levels were also low in HCCs of both noncirrhotic and cirrhotic livers, implying that VEGF-driven angiogenic sprouting accompanied by angiopoietin-driven vascular destabilization is not pronounced. In RCC, VEGF-A levels were one order of magnitude higher. At the same time, endothelially expressed Ang-2 was over 30-fold increased compared with expression in normal kidney, whereas Ang-1 expression was decreased. In hepatocellular carcinoma, tumor vascularization is not per se VEGF/angiopoietin driven. However, increased CD31 expression and morphological changes representative of sinusoidal capillarization in tumor vasculature indicate that vascular remodeling is taking place. This portends that therapeutic intervention of HCC at the level of the vasculature is optional, and that further studies into the molecular control thereof are warranted.


Blood stem cell-forming haemogenic endothelium in zebrafish derives from arterial endothelium.
Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4. Runx1-/- mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.


Application of the Gini correlation coefficient to infer regulatory relationships in transcriptome analysis.
One of the computational challenges in plant systems biology is to accurately infer transcriptional regulation relationships based on correlation analyses of gene expression patterns. Despite several correlation methods that are applied in biology to analyze microarray data, concerns regarding the compatibility of these methods with the gene expression data profiled by high-throughput RNA transcriptome sequencing (RNA-Seq) technology have been raised. These concerns are mainly due to the fact that the distribution of read counts in RNA-Seq experiments is different from that of fluorescence intensities in microarray experiments. Therefore, a comprehensive evaluation of the existing correlation methods and, if necessary, introduction of novel methods into biology is appropriate. In this study, we compared four existing correlation methods used in microarray analysis and one novel method called the Gini correlation coefficient on previously published microarray-based and sequencing-based gene expression data in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). The comparisons were performed on more than 11,000 regulatory relationships in Arabidopsis, including 8,929 pairs of transcription factors and target genes. Our analyses pinpointed the strengths and weaknesses of each method and indicated that the Gini correlation can compensate for the shortcomings of the Pearson correlation, the Spearman correlation, the Kendall correlation, and the Tukey's biweight correlation. The Gini correlation method, with the other four evaluated methods in this study, was implemented as an R package named rsgcc that can be utilized as an alternative option for biologists to perform clustering analyses of gene expression patterns or transcriptional network analyses.


Efficacy and tolerability of once-daily OROS hydromorphone and twice-daily extended-release oxycodone in patients with chronic, moderate to severe osteoarthritis pain: results of a 6-week, randomized, open-label, noninferiority analysis.
This study compared the efficacy and tolerability of a once-daily controlled-release formulation of hydromorphone (OROS) hydromorphone, Janssen-Cilag, Beerse, Belgium) and twice-daily extended-release (ER) oxycodone in patients with chronic, moderate to severe osteoarthritis (OA) pain. OROS hydromorphone is currently available only in Europe. Adults who met American College of Rheumatology clinical criteria for OA of the knee or hip with moderate to severe mean daily pain intensity despite chronic use of stable doses of NSAIDs or other nonsteroidal, nonopioid therapies were eligible for participation in this randomized, open-label study. The study consisted of a 14-day dose-titration and stabilization phase and a 28-day maintenance phase. OROS hydromorphone and ER oxycodone were initiated at dosages of 8 mg QD and 10 mg BID, respectively. Patients maintained diaries in which they rated their pain (from 0 = none to 3 = severe) and pain relief (from 0 = no relief to 4 = complete relief). Other assessments completed every 14 days included patient and investigator global evaluations of treatment effectiveness (scale from 1 = poor to 5 = excellent), the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index, and the Medical Outcomes Study (MOS) Sleep Scale. Adverse events (whether observed by study personnel, identified in response to questioning, or spontaneously reported) and vital signs were monitored throughout the study. The primary efficacy measures were the mean pain relief score at end point and the time from initiation of treatment to the third day of moderate to complete pain relief, as reported in the patient diary. Noninferiority analyses were conducted on all primary and secondary efficacy variables. One hundred thirty-eight patients (71 OROS hydromorphone, 67 ER oxycodone) received treatment (safety population), and 83 (60.1%) completed the study. Data from 124 patients were included in the efficacy analyses; the majority of these patients were white (85.5%) and female (69.4%), with a mean age of 63.6 years. The most commonly affected joint was the knee (79.8 %). At end point, the OROS hydromorphone group had a mean pain relief score of 2.3 (median, 2.0) and the ER oxycodone group had a mean pain relief score of 2.3 (median, 2.3) (95% CI, -0.30 to infinity). The mean time to the third day of moderate to complete pain relief was 6.2 days (median, 4.0) in the OROS hydromorphone group and 5.5 days (median, 5.0) in the ER oxycodone group (95% CI, -0.31 to infinity). Mean pain intensity decreased from baseline to end point by 0.6 point in the OROS hydromorphone group and by 0.4 point in the ER oxycodone group. Mean scores on the patient global evaluation improved by a respective 1.2 and 1.0 points (median, 1 in both groups). Approximately two thirds of patients in each group (67.2% and 66.7%) rated the overall effectiveness of treatment as good to excellent at end point. There were no statistically significant differences between groups in total WOMAC scores at end point, and similar improvements from baseline in the WOMAC physical function, stiffness, and pain scales were observed in both groups. Whereas MOS sleep outcomes scores improved from baseline in both groups, OROS hydromorphone was associated with a significantly greater improvement on the MOS Sleep Problems Index I compared with ER oxycodone (P < 0.045). Adverse events were comparable in both groups; the most frequently reported adverse events were nausea (35.2% and 29.9%), constipation (29.6% and 25.4%), somnolence (25.4% and 17.9%), vomiting (16.9% and 11.9%), and dizziness (14.1% and 22.4%). Adverse events led to study discontinuation in 35.2% (25/71) of patients in the OROS hydromorphone group and 32.8% (22/67) in the ER oxycodone group. Discontinuations due to adverse events during the titration phase were numerically greater in the OROS hydromorphone group (29.6% [21/71]) than in the ER oxycodone group (19.4% [13/67]). Only 1 serious adverse event (diarrhea in a patient receiving OROS hydromorphone) was considered possibly related to study drug. Once-daily OROS hydromorphone and twice-daily ER oxycodone provided similar pain relief in these patients with OA of the knee or hip. The tolerability profiles of the 2 agents were similar.


Single-cell transcriptomics using spliced leader PCR: Evidence for multiple losses of photosynthesis in polykrikoid dinoflagellates.
Most microbial eukaryotes are uncultivated and thus poorly suited to standard genomic techniques. This is the case for Polykrikos lebouriae, a dinoflagellate with ultrastructurally aberrant plastids. It has been suggested that these plastids stem from a novel symbiosis with either a diatom or haptophyte, but this hypothesis has been difficult to test as P. lebouriae dwells in marine sand rife with potential genetic contaminants. We applied spliced-leader targeted PCR (SLPCR) to obtain dinoflagellate-specific transcriptomes on single-cell isolates of P. lebouriae from marine sediments. Polykrikos lebouriae expressed nuclear-encoded photosynthetic genes that were characteristic of the peridinin-plastids of dinoflagellates, rather than those from a diatom of haptophyte. We confirmed these findings at the genomic level using multiple displacement amplification (MDA) to obtain a partial plastome of P. lebouriae. From these data, we infer that P. lebouriae has retained the peridinin plastids ancestral for dinoflagellates as a whole, while its closest relatives have lost photosynthesis multiple times independently. We discuss these losses with reference to mixotrophy in polykrikoid dinoflagellates. Our findings demonstrate new levels of variation associated with the peridinin plastids of dinoflagellates and the usefulness of SLPCR approaches on single cell isolates. Unlike other transcriptomic methods, SLPCR has taxonomic specificity, and can in principle be adapted to different splice-leader bearing groups.


An atypical Bacillus anthracis infection in a bull-A potential occupational health hazard.
Bacillus anthracis infecting cattle is usually identified based on the typical symptom: sudden death. Bacillus anthracis causing atypical symptoms may remain undiagnosed and represent a potential occupational health hazard for, that is veterinarians and producers, butchers and tanners. In the year 2004, one case of sudden death in a dairy farm in southern Finland was diagnosed as bovine anthrax. Four years later 2008, an atypical case of anthrax was diagnosed in the same holding. The bull was taken to the Production Animal Hospital of the Faculty of Veterinary Medicine, University of Helsinki because of fever, loss of appetite and a symmetrically swollen scrotal sac. Penicillin treatment cured the fever but not the swollen scrotum. Before the intended therapeutic castration, a punctuate consisting of 10 ml fluid collected into a syringe from the scrotal sac was cultivated on blood agar at 37°C. After 24 hr, an almost pure culture of a completely non-hemolytic Bacillus cereus-like bacteria was obtained. The strain was identified as B. anthracis using Ba-specific primers by the Finnish Food Safety Authority (RUOKAVIRASTO). After the diagnosis, the bull was euthanized and destroyed, the personnel were treated with prophylactic antibiotics and the clinic was disinfected. In this particular case, treatment with water, Virkon S and lime seemed to be effective to eliminate endospores and vegetative cells since no relapses of anthrax have occurred in 10 years. This case is the last reported anthrax case in Finland.


An investigation of the distal histidyl hydrogen bonds in oxyhemoglobin: effects of temperature, pH, and inositol hexaphosphate.
On the basis of X-ray crystal structures and electron paramagnetic resonance (EPR) measurements, it has been inferred that the O(2) binding to hemoglobin is stabilized by the hydrogen bonds between the oxygen ligands and the distal histidines. Our previous study by multinuclear nuclear magnetic resonance (NMR) spectroscopy has provided the first direct evidence of such H-bonds in human normal adult oxyhemoglobin (HbO(2) A) in solution. Here, the NMR spectra of uniformly (15)N-labeled recombinant human Hb A (rHb A) and five mutant rHbs in the oxy form have been studied under various experimental conditions of pH and temperature and also in the presence of an organic phosphate, inositol hexaphosphate (IHP). We have found significant effects of pH and temperature on the strength of the H-bond markers, i.e., the cross-peaks for the side chains of the two distal histidyl residues, α58His and β63His, which form H-bonds with the O(2) ligands. At lower pH and/or higher temperature, the side chains of the distal histidines appear to be more mobile, and the exchange with water molecules in the distal heme pockets is faster. These changes in the stability of the H-bonds with pH and temperature are consistent with the changes in the O(2) affinity of Hb as a function of pH and temperature and are clearly illustrated by our NMR experiments. Our NMR results have also confirmed that this H-bond in the β-chain is weaker than that in the α-chain and is more sensitive to changes in pH and temperature. IHP has only a minor effect on these H-bond markers compared to the effects of pH and temperature. These H-bonds are sensitive to mutations in the distal heme pockets but not affected directly by the mutations in the quaternary interfaces, i.e., α(1)β(1) and/or α(1)β(2) subunit interface. These findings provide new insights regarding the roles of temperature, hydrogen ion, and organic phosphate in modulating the structure and function of hemoglobin in solution.


Climatic conditions produce contrasting influences on demographic traits in a long-distance Arctic migrant.
The manner in which patterns of variation and interactions among demographic rates contribute to population growth rate (λ) is key to understanding how animal populations will respond to changing climatic conditions. Migratory species are likely to be particularly sensitive to climatic conditions as they experience a range of different environments throughout their annual cycle. However, few studies have provided fully integrated demographic analyses of migratory populations in response to changing climatic conditions. Here, we employed integrated population models to demonstrate that the environmental conditions experienced during a short but critical period play a central role in the demography of a long-distance migrant, the light-bellied Brent goose (Branta bernicla hrota). Female survival was positively associated with June North Atlantic Oscillation (NAO) values, whereas male survival was not. In contrast, breeding productivity was negatively associated with June NAO, suggesting a trade-off between female survival and reproductive success. Both adult female and adult male survival showed low temporal variation, whereas there was high temporal variation in recruitment and breeding productivity. In addition, while annual population growth was positively correlated with annual breeding productivity, a sensitivity analysis revealed that population growth was most sensitive to changes in adult survival. Our results demonstrate that the environmental conditions experienced during a relatively short-time window at the start of the breeding season play a critical role in shaping the demography of a long-distant Arctic migrant. Crucially, different demographic rates responded in opposing directions to climatic variation, emphasising the need for integrated analysis of multiple demographic traits when understanding population dynamics.


Changes in naïve and memory T-cells in elite swimmers during a winter training season.
High intensity training regimens appear to put athletes at a higher risk of illness. As these have been linked to alterations in the proportions of differentiated T cells, how training load affects these populations could have important implications for athlete susceptibility to disease. This study examined the effect of a winter training season on the proportions of circulating naïve and memory T cells subsets of high competitive level swimmers. Blood samples were taken at rest at 4 time-points during the season: before the start of the season (t0-September), after 7weeks of an initial period of gradually increasing training load (t1-November), after 6weeks of an intense training cycle (t2-February) and 48h after the main competition (t3-April) and from eleven non-athlete controls at 2 similar time-points (t2 and t3). CD4, CD8 and gamma-delta (γδ) T cells expressing the naïve (CCR7(+)CD45RA(+)), central-memory (CM-CCR7(+)CD45RA(-)), effector-memory (EM-CCR7(-)CD45RA(-)) and terminal effector (TEMRA-CCR7(-)CD45RA(+)) were quantified by flow cytometry. Statistical analyses were performed using multilevel modeling regression. Both T CD4(+) naïve and CM presented a linear increase in response to the first moment of training exposure, and had an exponential decrease until the end of the training exposure. As for TCD4(+) EM, changes were observed from t2 until the end of the training season with an exponential trend, while TCD4(+) TEMRA increased linearly throughout the season. TCD8(+) naïve increased at t1 and decreased exponentially thereafter. TCD8(+) TEMRA values decreased at t1 and increased exponentially until t3. γδT-EM had an increase at t1 and an exponential decrease afterwards. In contrast, γδT-TEMRA decreased at t1 and exponentially increased during the remaining 20weeks of training. An increase in TEMRA and EM T cells alongside a decrease in naïve T cells could leave athletes more susceptible to illness in response to variation in training stimulus during the season.


Cross Platform Standardisation of an Experimental Pipeline for Use in the Identification of Dysregulated Human Circulating MiRNAs.
Micro RNAs (miRNAs) are a class of highly conserved small non-coding RNAs that play an important part in the post-transcriptional regulation of gene expression. A substantial number of miRNAs have been proposed as biomarkers for diseases. While reverse transcriptase Real-time PCR (RT-qPCR) is considered the gold standard for the evaluation and validation of miRNA biomarkers, small RNA sequencing is now routinely being adopted for the identification of dysregulated miRNAs. However, in many cases where putative miRNA biomarkers are identified using small RNA sequencing, they are not substantiated when RT-qPCR is used for validation. To date, there is a lack of consensus regarding optimal methodologies for miRNA detection, quantification and standardisation when different platform technologies are used. In this study we present an experimental pipeline that takes into consideration sample collection, processing, enrichment, and the subsequent comparative analysis of circulating small ribonucleic acids using small RNA sequencing and RT-qPCR. Initially, a panel of miRNAs dysregulated in circulating blood from breast cancer patients compared to healthy women were identified using small RNA sequencing. MiR-320a was identified as the most dysregulated miRNA between the two female cohorts. Total RNA and enriched small RNA populations (<30 bp) isolated from peripheral blood from the same female cohort samples were then tested for using a miR-320a RT-qPCR assay. When total RNA was analysed with this miR-320a RT-qPCR assay, a 2.3-fold decrease in expression levels was observed between blood samples from healthy controls and breast cancer patients. However, upon enrichment for the small RNA population and subsequent analysis of miR-320a using RT-qPCR, its dysregulation in breast cancer patients was more pronounced with an 8.89-fold decrease in miR-320a expression. We propose that the experimental pipeline outlined could serve as a robust approach for the identification and validation of small RNA biomarkers for disease.


Brinzolamide Dimethyl Sulfoxide In Situ Gelling Ophthalmic Solution: Formulation Optimisation and In Vitro and In Vivo Evaluation.
In the present work, a cost-effective, stable and sustained release ophthalmic solution formulation of brinzolamide (BRZ) was developed for the treatment of glaucoma. The prototype formulation undergoes 'in situ gelling' when administered in the eye, thereby providing longer residence (16-24 h). As a result, the same therapeutic endpoint is achieved with once daily dosing vis-à-vis the commercially available product Azopt® (brinzolamide 1.0% w/v, Alcon Laboratories, USA) that requires 3-4 times instillations per day. The prototype formulations were prepared using dimethyl sulfoxide, polyoxyl 35 castor oil and polysorbate 80. Gellan gum was used as the in situ gelling agent. Formulation variables like (i) concentration of the drug, dimethyl sulfoxide and in situ gelling agent and (ii) type and concentration of solubiliser showed a significant effect on the solubility of brinzolamide, in vitro gelling time, in vitro drug release and in situ gel stability. Prototype formulations were evaluated in New Zealand white rabbits for ocular toxicity and efficacy study. The tested formulations were well tolerated and reduced intraocular pressure (IOP) from 25-28 to 12-14 mmHg compared to saline and placebo control samples. Additionally, a significant increase in the area under change in IOP from baseline (ΔIOP) vs. time curve and a longer mean residence time (MRT) were also observed for the test formulations (7.4 to 17.7 h) compared to the commercially available suspension of Azopt® (4.9 h) (p < 0.0001). Thus, 'in situ gelling' formulation strategy described in this work can work as a viable option for ocular delivery of brinzolamide for the treatment of glaucoma.


A multimethod approach examining the relative contributions of optimism and pessimism to cardiovascular disease risk markers.
Although dispositional optimism and pessimism are associated with cardiovascular disease (CVD), their relative independence and unique contributions to CVD risk are unclear. This study addressed these issues by using multiple indicators of optimism and pessimism and linking them to objective risk factors for CVD. A diverse sample of adults (N = 300) completed baseline assessments (including global reports of optimism and pessimism), a 2-day/1-night EMA protocol with ambulatory blood pressure (BP) at 45-min intervals, and had inflammatory markers and carotid intima media imaging collected. EMA reports of momentary positive and negative expectations were averaged to form intraindividual (person) means of optimism and pessimism, respectively. Optimism and pessimism were only modestly correlated between- and within-assessment methods. Higher pessimism, regardless of assessment method, predicted both lower odds of whether BP dipping occurred and a smaller degree of dipping, but was unrelated to other biomarkers. Optimism was not uniquely predictive of CVD risk factors. Pessimism thus appears to exhibit stronger relative contribution to risk indicators of CVD than optimism.


Immunoassay of specific IgE: low level assays require measurement of allergen specific assay background.
The allergen-specific backgrounds of a paper disk radioimmunoassay (RIA) system and a cellulose sponge fluorescent enzyme immunoassay (FEIA) system were evaluated using three inhalants, timothy, short ragweed, and cat, with reagents obtained from the same manufacturer. Radioimmunoassay was performed with Phadebas RAST reagents by the modified RAST method, and FEIA by the Pharmacia CAP System. Horse serum, 5% HSA, assay diluent, and serum pools from nonallergic and allergic patients, were assayed. The lower limit of detection (LLD) was defined using both the Z distribution (as is conventional) and the t distribution. The solid phases, analytes, and assays differed (p less than .001) in background results. For RIA, background was lowest for timothy and highest for cat; for FEIA, background was lowest for cat and highest for short ragweed. For RIA, background assessed with the allergic serum pool was higher than the other analytes; for FEIA, responses of the five analytes did not differ. For timothy and short ragweed, background of RIA was lower than FEIA. For FEIA, the highest LLD calculated using the Z distribution was 11 SD lower than the manufacturer's recommended quantitative cutoff; for RIA, the highest LLD calculated was 2.5 SD higher than the recommended analytic cutoff. The analytic false positive rate for RIA may differ between allergic and nonallergic patient populations. Laboratories reporting results near either assay's background should set LLD based on assay of allergen-specific negative controls in each assay run.


"We are like co-wives": Traditional healers' views on collaborating with the formal Child and Adolescent Mental Health System in Uganda.
Early identification and management of mental illness in childhood and adolescence helps to avert debilitating mental illness in adulthood but the attention given to Child and Adolescent Mental Health (CAMH) has until recently been low. Traditional healers are often consulted by patients with mental illness and in Uganda, up to 60% of patients attending traditional healers have moderate to severe mental illness. Poor access to CAMH care in Uganda creates a treatment gap that could be met through enhanced collaboration between traditional healers and biomedical health systems. The aim of this study was to explore traditional healers' views on their collaboration with biomedical health systems so as to inform the implementation of strategies to improve access to CAMH services in Uganda. In-depth interviews with 20 purposively selected traditional healers were conducted in November 2015. A semi-structured interview guide was used to explore: 1) The experiences of traditional healers with mental ill-health in children and adolescents; 2) their willingness to collaborate with the formal health system; and 3) their perception of clinicians' willingness to collaborate with them. Interviews were conducted in local languages and tape recorded. Data were analysed using thematic analysis. Traditional healers described several experiences managing children and adolescents with mental illness, which they ascribed to spiritual and physical causes. The spiritual explanations were a consequence of unhappy ancestral spirits, modern religions and witchcraft, while physical causes mentioned included substance abuse and fevers. No traditional healer had received a patient referred to them from a medical clinic although all had referred patients to clinics for non-mental health reasons. Traditional healers expressed distrust in biomedical health systems and believed their treatments were superior to medical therapies in alleviating mental suffering. They expressed willingness to collaborate with biomedical providers. However, traditional healers believe clinicians disregard them and would not be willing to collaborate with them. Potential for collaboration between traditional healers and biomedical health systems for improving access to CAMH services in Uganda exists, but is undermined by mutual mistrust and competition between traditional healers and clinicians.


Octreotide used for probing the type-II' β-turn CD and Raman markers.
Octreotide, a potent somatostatin (SST) analogue, is used as an antiproliferative drug in numerous endocrine tumors. Previous NMR investigations, basically performed in DMSO, had evidenced a type-II' β-turn structure for this cyclic peptide. However, apart a few incomplete studies by circular dichroism, a systematic analysis of the structural behavior of octreotide in aqueous solution as a function of concentration and ionic strength was still lacking. Here, we report the chemical synthesis and purification of octreotide for optical spectroscopic purposes accompanied by its structural analysis. Furthermore, we have used octreotide as a short size, well-defined model compound for analyzing the CD and Raman markers of a type-II' β-turn. CD data collected in the 25-250 μM range revealed the general trend of octreotide to undergo a disordered toward ordered structural transition upon increasing concentration. Especially, the β-turn CD markers could be characterized above 50 μM by a negative band at ~202 nm flanked by a shoulder at ~218 nm. On the basis of Raman spectra recorded as a function of concentration (1-20 mM), we could assign the markers at ~1678 and ~1650 cm(-1) in the amide I region, and at ~1303, ~1288, and ~1251 cm(-1) in the amide III region, to the type-II' β-turn structure. The stability of the intermolecular antiparallel β-sheet formed in octreotide could be confirmed by the rigidity of the disulfide bridge which adopts a preferential gauche-gauche-gauche rotamer along the -Cβ-S-S-Cβ- moiety of the linked cysteines. The present analysis permits a better understanding of the differences between the structural features of SST-14 and its routinely used analogue, octreotide.


Modern stem cell therapy: approach to disease.
Various types of stem cells exist, each with their own advantages and disadvantages. Considering the current available evidence, important preclinical and clinical studies regarding the therapeutic potential of stem cells, stem cell therapy might be the important strategy for tissue repair. The development of stem cell therapy for tissue repair has primarily relied on stem cells, especially mesenchymal stem cells. Multilineage differentiation into all of the described cells are considered as important candidates for a range of diseases like neurological diseases, cardiovascular diseases, gastrointestinal cancer and genetic defects, as well as for acute and chronic wounds healing and pharmaceutical treatment. We review the properties and multipotency of stem cells and their differentiation potential, once cultured under specific growth conditions, for use in cell-based therapies and functional tissue replacement.


Is the time between first diagnosis of paroxysmal atrial fibrillation and cryoballoon ablation a predictor of efficacy?
Cryoablation is an indicated therapy for the treatment of recurrent atrial fibrillation through pulmonary vein isolation; however, the optimal time between first diagnosis of atrial fibrillation and cryoablation is still unknown. We aimed to assess the clinical efficacy and safety of early versus later treatment of patients with paroxysmal atrial fibrillation by cryoablation. Five hundred and ten patients underwent atrial fibrillation cryoablation and were prospectively followed for at least 6 months in 43 Italian cardiology centers. The population was divided into two groups according to the time since the first diagnosis of atrial fibrillation until the index cryoablation procedure. An early-treatment group had an elapsed time of 15 months or less from atrial fibrillation diagnosis until cryoablation, and the late-treatment group had an elapsed time of greater than 15 months. During the evaluation, clinical efficacy was defined as atrial fibrillation recurrence outside a landmark 90-day blanking period, and safety was defined as the reporting of all procedure-related complications. In the total cohort, cryoablation was performed after a median of 36 months from the point of the patient diagnosis with drug refractory symptomatic recurrent atrial fibrillation. The early-treatment group was composed of 130 (25%) patients, whereas the late-treatment group had 380 (75%) patients. Both cohorts had similar baseline clinical characteristics. Of 510 patients, 22 had a complication related to the procedure with no difference between the two groups. Multivariable analysis showed that the risk of atrial fibrillation recurrence was significantly higher in the late-treatment group (hazard ratio: 1.77; 95% confidence interval 1.00-3.13) CONCLUSION: In our multicenter observational examination, cryoablation was well tolerated and effective in the treatment of patients with drug refractory symptomatic paroxysmal atrial fibrillation. Reducing the time between diagnosis and ablation brought about a treatment that had a lower risk of atrial fibrillation recurrence with no change in safety.(Italian ClinicalService Project: NCT01007474).


Laparoendoscopic single-site nephroureterectomy for upper urinary tract urothelial carcinoma: outcomes of an international multi-institutional study of 101 patients.
To report a large multi-institutional series of laparoendoscopic single-site (LESS) nephroureterectomy (NU). Data on all cases of LESS-NU performed between 2008 and 2012 at 15 institutions were retrospectively gathered. The main demographic data and perioperative outcomes were analysed. The study included 101 patients whose mean (SD) age was 66.4 (9.9) years and mean (SD) body mass index was 24.8 (4) kg/m², and of whom 29.7% had undergone previous abdominal/pelvic surgery. The mean (SD) operating time was 221.4 (73.7) min, estimated blood loss 231.7 (348.0) mL. A robot-assisted LESS technique was applied in 25.7% of cases. An extra trocar was inserted in 28.7% of cases to complete the procedure. Conversion to open surgery was necessary in three cases (3.0%). There was no bladder cuff excision in 20.8% of cases, and excision was carried out using a variety of techniques in the remaining cases. Six intra-operative complications occurred (5.9%). The mean (SD) length of hospital stay was 6.3 (3.5) days. The overall postoperative complication rate was 10.0%, and most of the complications were low grade (Clavien grades 1 and 2). The mean tumour size was 3.1 (1.9) cm. Pathological staging was pTis in two patients, pTa in 12 patients, pT1 in 42 patients, pT2 in 20 patients, pT3 in 23 patients and pT4 in two patients. Pathological grade was high in 71 and low in 30 patients. At a mean follow-up of 14 months, six patients (5.9%) had died. Disease recurrence (including distant and bladder recurrence) was detected in 22.8% of patients, with a mean time to recurrence of 11.5 months. This study reports the largest multi-institutional experience of LESS-NU to date. Peri-operative outcomes mirror those of published standard laparoscopy series. Despite encouraging early findings, longer follow-up is needed to determine the oncological efficacy of the procedure.


Evaluation of the living with hope program for rural women caregivers of persons with advanced cancer.
Hope has been identified as a key psychosocial resource among family caregivers to manage and deal with the caregiver experience. The Living with Hope Program is a self-administered intervention that consists of watching an international award winning Living with Hope film and participating in a two week hope activity ("Stories of the Present"). The purpose of this study was to examine the effects of the Living with Hope Program on self-efficacy [General Self-Efficacy Scale], loss and grief [Non-Death Revised Grief Experience Inventory], hope [Herth Hope Index] and quality of life [Short-Form 12 version 2 (SF-12v2)] in rural women caring for persons with advanced cancer and to model potential mechanisms through which changes occurred. A time-series embedded mixed method design was used, with quantitative baseline outcome measures repeated at day 7, day 14, and 3, 6 and 12 months. Qualitative data from the hope activity informed the quantitative data. Thirty-six participants agreed to participate with 22 completing all data collection. General estimating equations were used to analyze the data. Herth Hope Index scores (p=0.05) had increased significantly from baseline at day 7. General Self Efficacy Scale scores were significantly higher than baseline at all data time points. To determine the mechanisms of the Living with Hope Program through which changes occurred, results of the data analysis suggested that as General Self Efficacy Scale scores increased (p<0.001) and Non-death Revised Grief Experience Inventory scores decreased (p=0.01) Herth Hope Index scores increased. In addition as Herth Hope Index scores increased (p<0.001) and Non-death Revised Grief Experience Inventory scores decreased (p=0.01), SF-12v2 mental health summary scores increased. Qualitative data suggested that through the hope activity (Stories of the Present) the participants were able to find positives and hope in their experience. The Living with Hope Program has potential to increase hope and improve quality of life for rural women caregivers of persons with advanced cancer. The possible mechanisms by which changes in hope and quality of life occur are by decreasing loss and grief and increasing self-efficacy. Registration ClinicalTrials.gov, NCT01081301.


A case study of nitrogen saturation in western U.S. forests.
Virtually complete nitrification of the available ammonium in soil and nitrification activity in the forest floor are important factors predisposing forests in the San Bernardino Mountains of southern California to nitrogen (N) saturation. As a result, inorganic N in the soil solution is dominated by nitrate. High nitrification rates also generate elevated nitric oxide (NO) emissions from soil. High-base cation saturation of these soils means that soil calcium depletion or effects associated with soil acidification are not an immediate risk for forest health as has been postulated for mesic forests in the eastern U.S. Physiological disturbance (e.g., altered carbon [C] cycling, reduced fine root biomass, premature needle abscission) of ozone-sensitive ponderosa pine trees exposed to high N deposition and high ozone levels appear to be the greater threat to forest sustainability. However, N deposition appears to offset the aboveground growth depression effects of ozone exposure. High nitrification activity reported for many western ecosystems suggests that with chronic N inputs these systems are prone to N saturation and hydrologic and gaseous losses of N. High runoff during the winter wet season in California forests under a Mediterranean climate may further predispose these watersheds to high nitrate leachate losses. After 4 years of N fertilization at a severely N saturated site in the San Bernardino Mountains, bole growth unexpectedly increased. Reduced C allocation below- ground at this site, presumably in response to ozone or N or both pollutants, may enhance the bole growth response to added N.


Response of an actin filament network model under cyclic stretching through a coarse grained Monte Carlo approach.
Cells are complex, dynamic systems that actively adapt to various stimuli including mechanical alterations. Central to understanding cellular response to mechanical stimulation is the organization of the cytoskeleton and its actin filament network. In this manuscript, we present a minimalistic network Monte Carlo based approach to model actin filament organization under cyclic stretching. Utilizing a coarse-grained model, a filament network is prescribed within a two-dimensional circular space through nodal connections. When cyclically stretched, the model demonstrates that a perpendicular alignment of the filaments to the direction of stretch emerges in response to nodal repositioning to minimize net nodal forces from filament stress states. In addition, the filaments in the network rearrange and redistribute themselves to reduce the overall stress by decreasing their individual stresses. In parallel, we cyclically stretch NIH 3T3 fibroblasts and find a similar cytoskeletal response. With this work, we test the hypothesis that a first-principles mechanical model of filament assembly in a confined space is by itself capable of yielding the remodeling behavior observed experimentally. Identifying minimal mechanisms sufficient to reproduce mechanical influences on cellular structure has important implications in a diversity of fields, including biology, physics, medicine, computer science, and engineering.


Naloxone increases the frequency of pulsatile luteinizing hormone secretion in women with hyperprolactinemia.
The ability to change the frequency and amplitude of pulsatile GnRH secretion may be an important mechanism in maintaining regular ovulatory cycles. Hyperprolactinemia is associated with anovulation and slow frequency LH (GnRH) secretion in women. To assess whether the slow frequency of LH (GnRH) secretion is due to increased opioid activity, we examined the effect of naloxone infusions in eight amenorrheic hyperprolactinemic women (mean +/- SE, serum PRL, 160 +/- 59 micrograms/L). After a baseline period, either saline or naloxone was infused for 8 h on separate days, and LH was measured in blood obtained at 15-min intervals. Additional samples were obtained for plasma FSH, PRL, estradiol, and progesterone. Responses to exogenous GnRH were assessed at the end of the infusions. LH pulse frequency increased in all subjects from a mean of 4.0 +/- 0.5 pulses/10 h (mean +/- SE) during saline infusion to 8.0 +/- 1.0 pulses/10 h during naloxone infusion (P less than 0.01). LH pulse amplitude did not change, and mean plasma LH increased from 7.4 +/- 0.8 IU/L (+/- SE) to 11.2 +/- 1.5 IU/L during naloxone (P less than 0.01). A small but significant increase was seen in mean plasma FSH. Plasma PRL, estradiol, and progesterone were unchanged by naloxone infusion. These data suggest that elevated serum PRL reduces the frequency of LH (GnRH) secretion by increasing hypothalamic opioid activity and suggest that the anovulation in hyperprolactinemia is consequent upon persistent slow frequency LH (GnRH) secretion.


Direct quantitation of omega-3 fatty acid intake of Canadian residents of a long-term care facility.
An increased dietary intake of n-3 highly unsaturated fatty acids (HUFA; >or=20 carbons, >or=3 carbon-carbon double bonds), particularly eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), is associated with the decreased risk and incidence of several morbidities afflicting the elderly, including cognitive decline, dementia, rheumatoid arthritis, and macular degeneration. In this study, the dietary intake and blood levels of fatty acids were directly determined in residents of a retirement home or assisted living phase of a continuum of care facility for Canadian seniors. Finger-tip-prick blood samples, 3-day food duplicates, and 3-day food records were collected. The fatty acid composition of food duplicates and blood was determined by gas chromatography. Fifteen participants (7 male, 8 female; 87.1 +/- 4.8 years of age) completed the protocol. The daily intake of EPA and DHA combined, determined directly, was 70 mg (95% CI, 41-119) or 0.036% of total energy (95% CI, 0.022-0.058). In finger-tip-prick blood, the percent of n-3 HUFA in total HUFA of whole blood, a biomarker of n-3 polyunsaturated fatty acid status, was 28.8 +/- 5.2%. Correlations between daily n-3 HUFA intake and n-3 HUFA in blood were not significant (r = 0.14; n = 15), but became significant after the removal of 2 participants who appeared to consume fish irregularly (r = 0.59; n = 13). The n-3 HUFA intake and corresponding n-3 HUFA blood levels of Canadian long-term care residents are lower than levels estimated to prevent several morbidities associated with aging.


Specificity and functional activity of anti-Burkholderia pseudomallei polysaccharide antibodies.
The lipopolysaccharide (LPS) of Burkholderia pseudomallei, the causative agent of melioidosis, consists of two O-antigenic polysaccharides designated O-PS I and O-PS II. In this study, the O-PS specificity and functional activity of a protective polyclonal antiserum and an immunoglobulin M (IgM) monoclonal antibody were determined. The polyclonal antiserum recognized both O-PS I and O-PS II, while the monoclonal antibody was O-PS II specific. Both mediated phagocytic killing of B. pseudomallei by polymorphonuclear leukocytes. Patients acutely infected with B. pseudomallei also produced antibodies to the two O-PSs, but these antibodies were not produced by asymptomatic individuals from an area of endemicity who were seropositive by an indirect hemagglutination test using sonicated heat-killed whole organisms as antigen. IgM antibodies were detected only in patients with localized infection. IgG antibodies were detected in all acutely infected patients, but there was no significant difference in antibody levels among patients with localized infection, patients who survived septicemic illness, and patients who died from septicemic illness. Further analysis of the IgG response revealed production of IgG1 and IgG2 antibodies by all patient groups, while an IgG3 response was seen only in survivors of septicemic infection. IgG4 was not detectable even when a fivefold-lower serum dilution was used. Patient sera also mediated phagocytic killing by polymorphonuclear leukocytes, and the killing effect was enhanced by complement. These results suggest that antibodies to the LPS O-polysaccharides of B. pseudomallei are protective by promoting phagocytic killing. The antibodies develop during human infection and may facilitate clearance of the organisms, as seen in a diabetic rat model of B. pseudomallei infection.


Osteopathic manipulative treatment applications for the emergency department patient.
The emergency department (ED) setting offers osteopathic physicians multiple opportunities to provide osteopathic manipulative treatment (OMT) as either the primary therapy or as an adjunct to the intervention. In doing so, osteopathic physicians can decrease or eliminate the morbidity and symptoms associated with protracted dysfunction. Low back pain, chest pain, torticollis, asthma, and sinusitis are some of the illnesses in which OMT should be implemented as part of the management plan, note the authors. They provide a guide to the general usage of OMT when the aforementioned illnesses present themselves in ED patients, but also emphasize the importance of first ruling out any underlying illnesses that could be manifested by musculoskeletal symptoms.


The Genomic Basis of Evolutionary Innovation in Pseudomonas aeruginosa.
Novel traits play a key role in evolution, but their origins remain poorly understood. Here we address this problem by using experimental evolution to study bacterial innovation in real time. We allowed 380 populations of Pseudomonas aeruginosa to adapt to 95 different carbon sources that challenged bacteria with either evolving novel metabolic traits or optimizing existing traits. Whole genome sequencing of more than 80 clones revealed profound differences in the genetic basis of innovation and optimization. Innovation was associated with the rapid acquisition of mutations in genes involved in transcription and metabolism. Mutations in pre-existing duplicate genes in the P. aeruginosa genome were common during innovation, but not optimization. These duplicate genes may have been acquired by P. aeruginosa due to either spontaneous gene amplification or horizontal gene transfer. High throughput phenotype assays revealed that novelty was associated with increased pleiotropic costs that are likely to constrain innovation. However, mutations in duplicate genes with close homologs in the P. aeruginosa genome were associated with low pleiotropic costs compared to mutations in duplicate genes with distant homologs in the P. aeruginosa genome, suggesting that functional redundancy between duplicates facilitates innovation by buffering pleiotropic costs.


Resolution of orbitocerebral aspergillosis during combination treatment with voriconazole and amphotericin plus adjunctive cytokine therapy.
Orbitocerebral aspergillosis has a very high fatality rate and cure is unusual. We describe the successful management of a child with cereberal aspergillosis who had a dramatic response to therapy with a combination of liposomal amphotericin and voriconazole with adjunctive cytokine therapy during immunosuppresive chemotherapy for acute lymphoblastic leukaemia.


General anaesthesia in elderly patients with cardiovascular disorders: choice of anaesthetic agent.
Our population is aging; currently 15% of the Western population are aged >65 years, and represent 25% of those undergoing surgery. The proportion of the population aged >or=65 years is rapidly growing, and an increasing number are affected with cardiovascular disease. The older person is a high-risk patient. This is because of their altered physiology and associated co-morbidities, as well as the pharmacokinetic and pharmacodynamic changes that may alter drug responses. There is considerable variability seen in the physical and physiological states of individual patients within the older population. This has an important impact on choosing a safe anaesthetic technique for each individual, which in turn can influence the morbidity and mortality in this population. The physiological changes in the aging cardiovascular system affect the arterial and venous vasculature, myocardium and autonomic nervous system, making the older person more prone to cardiovascular instability. In addition to the physiological changes, the cardiovascular status of the older person tends to be compromised by associated pathological conditions that are more common with increasing age. Pharmacokinetic and pharmacodynamic changes must be taken into account when deciding about drug dosing in this age group. Aspects of dose reduction, titration of drugs, dosing intervals and the pharmacodynamic effects of each class of drug are explained in detail in the text. The major challenge in anaesthesia for the older person with cardiovascular disease is maintenance of haemodynamic stability, particularly in the face of reduced physiological reserve and capability to respond to periods of instability. An appropriate anaesthetic technique must be selected to minimize haemodynamic changes and maintain near normal physiological status. The other key objective is to minimize the incidence of adverse outcomes, such as perioperative myocardial ischaemia/infarction, arrhythmias, heart failure, postoperative cognitive dysfunction and stroke. No single anaesthetic regimen or agent can be advocated. Knowledge of the pharmacokinetic and pharmacodynamic principles of anaesthetic agents and their altered response in elderly patients is essential when selecting an anaesthetic agent. This article provides a practical guide to the selection and use of general anaesthetic agents in older patients with cardiovascular disorders, highlighting the differences among various agents.


Common intestinal parasites.
Intestinal parasites cause significant morbidity and mortality. Diseases caused by Enterobius vermicularis, Giardia lamblia, Ancylostoma duodenale, Necator americanus, and Entamoeba histolytica occur in the United States. E. vermicularis, or pinworm, causes irritation and sleep disturbances. Diagnosis can be made using the "cellophane tape test." Treatment includes mebendazole and household sanitation. Giardia causes nausea, vomiting, malabsorption, diarrhea, and weight loss. Stool ova and parasite studies are diagnostic. Treatment includes metronidazole. Sewage treatment, proper handwashing, and consumption of bottled water can be preventive. A. duodenale and N. americanus are hookworms that cause blood loss, anemia, pica, and wasting. Finding eggs in the feces is diagnostic. Treatments include albendazole, mebendazole, pyrantel pamoate, iron supplementation, and blood transfusion. Preventive measures include wearing shoes and treating sewage. E. histolytica can cause intestinal ulcerations, bloody diarrhea, weight loss, fever, gastrointestinal obstruction, and peritonitis. Amebas can cause abscesses in the liver that may rupture into the pleural space, peritoneum, or pericardium. Stool and serologic assays, biopsy, barium studies, and liver imaging have diagnostic merit. Therapy includes luminal and tissue amebicides to attack both life-cycle stages. Metronidazole, chloroquine, and aspiration are treatments for liver abscess. Careful sanitation and use of peeled foods and bottled water are preventive.


Strategies for breastfeeding success.
Breastfeeding provides significant health benefits for infants and mothers. However, the United States continues to fall short of the breastfeeding goals set by the Healthy People 2010 initiative. The American Academy of Family Physicians, the American Academy of Pediatrics, and the American College of Obstetrics and Gynecology have policy statements supporting breastfeeding that reflect recent advancements in understanding the mechanisms underlying the benefits of breastfeeding and in the clinical management of breastfeeding. Despite popular belief, there are few contraindications to breastfeeding. Providing maternal support and structured antenatal and postpartum breastfeeding education are the most effective means of achieving breastfeeding success. In addition, immediate skin-to-skin contact between mother and infant and early initiation of breastfeeding are shown to improve breastfeeding outcomes. When concerns about lactation arise during newborn visits, the infant must be carefully assessed for jaundice, weight loss, and signs of failure to thrive. If a work-up is required, parents should be supported in their decision to breastfeed. Certified lactation consultants can provide valuable support and education to patients. Physicians should educate working women who breastfeed about the availability of breast pumps and the proper storage of expressed breast milk. Physicians must be aware of their patients' lactation status when prescribing medications, as some may affect milk supply or be unsafe for breastfeeding infants. Through support and encouragement of breastfeeding, national breastfeeding goals can be met.


Fast radiative transfer model for simulation of infrared atmospheric sounding interferometer radiances.
A fast radiative transfer model has been developed for prelaunch simulation studies of Infrared Atmospheric Sounding Interferometer (IASI) data and for the exploitation of IASI radiances within the framework of a numerical weather prediction variational analysis scheme. The model uses profile-dependent predictors to parameterize the atmospheric optical depths and is fast enough to cope with the processing of observations in near real time and with the several thousands of transmittance calculations required to simulate radiances from a full range of atmospheric conditions. The development of the model has involved the selection of a training set of atmospheric profiles, the production of a line-by-line transmittance database, the selection of optimal predictors for the gases considered in the study, and the production of regression coefficients for the fast transmittance scheme. The model fit to the line-by-line radiances shows that it can reproduce the line-by-line radiances to a degree of accuracy that is at or below the instrumental noise.