Mitochondrial (Dys)function and Insulin Resistance: From Pathophysiological Molecular Mechanisms to the Impact of Diet.
Mitochondrial dysfunction has been implicated in the pathogenesis of insulin resistance, the hallmark of type 2 diabetes mellitus (T2DM). However, the cause-effect relationship remains to be fully elucidated. Compelling evidence suggests that boosting mitochondrial function may represent a valuable therapeutic tool to improve insulin sensitivity. Mitochondria are highly dynamic organelles, which adapt to short- and long-term metabolic perturbations by undergoing fusion and fission cycles, spatial rearrangement of the electron transport chain complexes into supercomplexes and biogenesis governed by peroxisome proliferator-activated receptor γ co-activator 1α (PGC 1α). However, these processes appear to be dysregulated in type 2 diabetic individuals. Herein, we describe the mechanistic link between mitochondrial dysfunction and insulin resistance in skeletal muscle alongside the intracellular pathways orchestrating mitochondrial bioenergetics. We then review current evidence on nutritional tools, including fatty acids, amino acids, caloric restriction and food bioactive derivatives, which may enhance insulin sensitivity by therapeutically targeting mitochondrial function and biogenesis.


Fra-1 and c-Fos N-Terminal Deletion Mutants Impair Breast Tumor Cell Proliferation by Blocking Lipid Synthesis Activation.
Tumor cells require high rates of lipid synthesis to support membrane biogenesis for their exacerbated growth. The only two proteins known that activate phospholipid synthesis are Fra-1 and c-Fos, two members of the AP-1 family of transcription factors. These proteins that are overexpressed in human breast malignant tumors increase the rate of phospholipid synthesis at the endoplasmic reticulum through a mechanism independent of their nuclear function. The aim of this study was to inhibit breast tumor cell proliferation by modulating c-Fos and Fra-1 and regulate membrane biogenesis by controlling lipid synthesis rates. The molecular mechanism by which Fra-1 and c-Fos activate phospholipid synthesis was examined. Both proteins physically associate with the rate limiting enzyme CDP-DAG synthase through their N-terminus domain and activate it through their basic domain; neither protein associates to or activates the enzyme phosphatidylinositol synthase as determined through in vitro enzymatic reactions and FRET experiments. The N-terminus domain of both proteins act as negative dominant peptides that physically associate with CDP-DAG synthase but do not activate it. Proliferation of MDA-MB231 and 4T1 cells was impaired in vitro after inducing them to proliferate in the presence of the negative dominant peptides derived from Fra-1 and c-Fos. When tumors generated in Balb/c mice with the breast tumor cell line 4T1 were treated with these negative dominant peptides, a significant reduction in tumor growth was observed. Consequently, these Fra-1 and c-Fos negative dominant peptides can be exploited as a new therapeutic strategy to impair breast tumor cell proliferation.


Factors associated with intestinal perforation in children's non-typhi Salmonella toxic megacolon.
To evaluate the risk factors for intestinal perforation in children with toxic megacolon caused by non-typhi Salmonella infection. During an 11-year period we reviewed the records of children treated for non-typhi Salmonella infection. All of the subjects had positive stool culture for non-typhi Salmonella and were treated with intravenous ceftriaxone during hospitalization. Clinical data reviewed included demographic features, clinical manifestations, laboratory findings, radiologic findings, microbiology, therapeutic effect of hydration and rectal tube placement and the operative findings. Patients with toxic megacolon were defined as those having toxic appearance, diarrhea, high fever (>39 degrees C) and marked colon dilatation with maximal diameter > 1.5 times the width of the vertebra body of the first lumbar spine (L1-VB). To define the risk factors for patients with toxic megacolon complicated by intestinal perforation, patients were divided into two groups for analysis: P group, those complicated with intestinal perforation; and NP group, those without intestinal perforation. Differences in age, sex, severity of diarrhea, duration of fever, hemogram and its differential, culture, stool analysis, serum C-reactive protein (CRP), electrolytes, maximal colon diameter, medical therapy and timing of rectal tube insertion between the two groups were analyzed. Statistical analyses were conducted with chi square tests and multiple logistic regression. A total of 75 patients (P group, 27 patients; NP group, 48 patients) ages 4 months to 6 years were evaluated. With chi square analysis 7 variables were found to be significantly associated with intestinal perforation: age >1 year; fever >5 days; ratio of immature to total neutrophils >20%; serum CRP >200 mg/l; colon diameter >2.5 times the width of L1-VB; inadequate early hydration; and delay in rectal tube insertion. With multivariate analysis age >1 year, serum CRP >200 mg/l and colon diameter >2.5 times of width of L1-VB, inadequate early hydration and delay in rectal tube insertion were the most significant factors associated with intestinal perforation. Identification of patients with toxic megacolon associated with non-typhi Salmonella infection at risk for further intestinal perforation is possible. Early effective fluid resuscitation and rectal tube insertion may be helpful to prevent the occurrence of intestinal perforation.


Comparative modelling of cytochromes P450.
The superfamily of enzymes known as the cytochromes P450 (P450s) comprises a wide-ranging class of proteins with diverse functions. They are known, amongst other things, to be involved in the hormonal regulation of metabolism and reproduction, as well as having a major clinical significance through their association with diseases such as cancer, diabetes and hepatitis. Knowledge of the three-dimensional (3D) structure of a protein gives insight into its function. The 3D structures of P450s are therefore of considerable scientific interest. A number of high-resolution structures of P450s have been determined by X-ray crystallography and studies of these structures have provided valuable insights into the mechanism of these enzymes. Only one of these structures is mammalian and as yet there is no structural information on human P450s in the public domain. Until such a structure is solved it is necessary to employ alternative methods to gain structural insight into how human P450s perform their biological function. Here we report on the use of comparative modelling to predict the structure of human P450s based on knowledge of their amino acid sequences plus the 3D structures of other (not human) P450s. As an illustrative example of these techniques we have modelled the structure of P450 2C5 using five bacterial P450 structures as templates. We examine the importance of selecting suitable templates, obtaining a good amino acid sequence alignment, and evaluating the models generated. To improve the quality of the models an iterative cycle of sequence alignment, model building, and model evaluation is employed. The result is a model with excellent stereochemistry, good amino acid side chain environment properties, and a Calpha trace similar to the crystal structure.


Metabolic Profiling of High Egg Consumption and the Associated Lower Risk of Type 2 Diabetes in Middle-Aged Finnish Men.
Higher egg intake was previously associated with a lower risk of developing type 2 diabetes (T2D) in the prospective, population-based Kuopio Ischaemic Heart Disease Risk Factor Study (KIHD) in eastern Finland. Potential compounds that can explain this association are explored using nontargeted LC-MS-based metabolic profiling. Two hundred and thirty-nine baseline serum samples from the KIHD are analyzed in four groups: subjects with higher (mean intake one egg per day) or lower (mean intake two eggs per week) egg intake who developed T2D (cases) or remained heatlhy (controls) during the mean follow-up of 19.3 years. Different serum profiles of subjects who had either higher or lower egg intakes, and of those who developed type 2 diabetes or remained healthy, are observed. The higher baseline tyrosine level predicts higher odds of T2D (OR 1.94; 95% CI 1.45, 2.60; p < 0.001; FDR 0.023) along with an unknown hexose-containing compound (OR 2.13; 95% CI 1.57, 2.88; p < 0.001; FDR 0.005). Certain predominant metabolites in T2D cases are correlated positively with ones in the lower-egg-intake group and negatively with ones in the higher-egg-intake group. Our current findings may underline some potential metabolites that can explain how egg intake is associated with a lower risk of T2D.


A common polymorphism in the CYP3A7 gene is associated with a nearly 50% reduction in serum dehydroepiandrosterone sulfate levels.
CYP3A7, expressed in the human fetal liver and normally silenced after birth, plays a major role in the 16alpha-hydroxylation of dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and estrone. Due to a replacement of part of the CYP3A7 promoter with a sequence identical with the same region in the CYP3A4 promoter (referred to as CYP3A7*1C), some individuals still express a variant of the CYP3A7 gene later in life. The objective of this study was to examine the effect of the CYP3A7*1C polymorphism on serum steroid hormone levels. Two population-based cohort studies were performed. Study group 1 consisted of 208 subjects randomly selected from the Rotterdam Study, and study group 2 consisted of 345 elderly independently living men. Serum DHEA(S), androstenedione, estradiol, estrone, and testosterone levels were the main outcome measures. In study groups 1 and 2, heterozygous CYP3A7*1C carriers had almost 50% lower DHEAS levels compared with homozygous carriers of the reference allele [study group 1, 1.74 +/- 0.25 vs. 3.33 +/- 0.15 micromol/liter (P = 0.02); study group 2, 2.09 +/- 0.08 vs. 1.08 +/- 0.12 micromol/liter (P < 0.001)]. No differences in circulating DHEA, androstenedione, estradiol, or testosterone levels were found. However, in study group 2, serum estrone levels were lower in heterozygous CYP3A7*1C carriers compared with homozygous carriers of the reference allele (0.11 +/- 0.002 vs. 0.08 +/- 0.006 nmol/liter; P < 0.001). The CYP3A7*1C polymorphism causes the persistence of enzymatic activity of CYP3A7 during adult life, resulting in lower circulating DHEAS and estrone levels.


Metformin, Lifestyle Intervention, and Cognition in the Diabetes Prevention Program Outcomes Study.
We examined the association of the Diabetes Prevention Program (DPP) intervention arms (lifestyle intervention, metformin, and placebo) with cognition in the Diabetes Prevention Program Outcomes Study (DPPOS). We also examined metformin use, incident type 2 diabetes, and glycemia as exposures. The DPP lasted 2.8 years, followed by a 13-month bridge to DPPOS. Cognition was assessed in DPPOS years 8 and 10 (12 and 14 years after randomization) with the Spanish English Verbal Learning Test (SEVLT), letter fluency and animal fluency tests, Digit Symbol Substitution Test (DSST), and a composite cognitive score. A total of 2,280 participants (749 lifestyle, 776 metformin, and 755 placebo) aged 63.1 ± 10.7 years underwent cognitive assessments; 67.7% women, 54.6% non-Hispanic white, 20.7% non-Hispanic black, 14.6% Hispanic, 5.5% American Indian, and 4.6% Asian; 26.6% were homozygous or heterozygous for APOE-ε4. At the time of cognitive assessment, type 2 diabetes was higher in the placebo group (57.9%; P < 0.001) compared with lifestyle (47.0%) and metformin (50.4%). Metformin exposure was higher in the metformin group (8.72 years; P < 0.001) compared with placebo (1.43 years) and lifestyle (0.96 years). There were no differences in cognition across intervention arms. Type 2 diabetes was not related to cognition, but higher glycated hemoglobin at year 8 was related to worse cognition after confounder adjustment. Cumulative metformin exposure was not related to cognition. Exposure to intensive lifestyle intervention or metformin was not related to cognition among DPPOS participants. Higher glycemia was related to worse cognitive performance. Metformin seemed cognitively safe among DPPOS participants.


Increased plasma soluble uPAR level is a risk marker of respiratory cancer in initially cancer-free individuals.
Soluble urokinase plasminogen activator receptor (suPAR) is a stable plasma biomarker associated with inflammation and disease. This study tested the association between suPAR levels and incident respiratory, gastrointestinal, or other types of cancer in initially cancer-free individuals from a general population-based prospective study. Baseline plasma samples, baseline characteristics, and follow-up data were available from 2,656 individuals from the population-based Danish MONICA10 (MONItoring trends and determinants of CArdiovascular disease) study, followed for a median of 12.6 years. Cancer was diagnosed according to international classification of diseases (ICD) 8 and ICD-10 codes and suPAR levels were measured using a commercially available ELISA. The association of suPAR levels with incident cancer during follow-up was analyzed using Cox regression, adjusted for established risk factors and the inflammatory markers C-reactive protein (CRP) and leukocyte numbers. suPAR levels ranged from 0.6 to 22 ng/mL and median suPAR level was 4.01 ng/mL. An increase of 1 ng/mL in baseline suPAR was associated with adjusted HR of 1.61 (95% CI: 1.23-2.11, P < 0.001), 0.92 (95% CI: 0.69-1.24, P = 0.59), and 1.33 (95% CI: 1.13-1.58, P < 0.001) of being diagnosed with respiratory, gastrointestinal, and other cancer types, respectively. Elevated suPAR levels were associated with increased risk of incident respiratory cancer and other types of cancer, but not gastrointestinal cancers, independently of established risk factors, CRP, and leukocyte numbers. These findings suggest that inflammation is involved in cancer development. Risk algorithms based on established risk factors and risk-associated biomarkers should be developed and evaluated in large, general population-based studies. We suggest suPAR as a candidate for evaluation in cancer risk algorithms.


Dense intramembranous deposit disease: a clinical comparison of histological subtypes.
The accumulation of osmiophilic dense deposits in glomerular mesangial and basement membranes (dense intramembranous deposit disease, or DIDD) is associated with variable histologic alterations of the kidneys. We compared clinical features and long-term renal outcome in 21 patients representing two histologic subtypes of DIDD, namely membranoproliferative glomerulonephritis (MPGN) and focal segmental glomerulonephritis (FSGN). We found that MPGN-type DIDD in 12 patients was associated with nephrotic syndrome in 12, persistent hypocomplementemia in 10 and progression to chronic renal insufficiency in 8. In 9 patients with the focal segmental variety of DIDD, nephrotic syndrome was observed in 3, persistent hypocomplementemia in none, and progression to renal insufficiency in 2 (significance: nephrotic syndrome, p = 0.001; persistent hypocomplementemia, p = 0.0001; chronic renal insufficiency, p = 0.02). In one patient transition from focal segmental to MPGN-type DIDD was observed. We conclude that DIDD is a heterogeneous disorder, and that certain clinical and histologic features may be useful in predicting ultimate outcome.


Regulation of synaptic extracellular matrix composition is critical for proper synapse morphology.
Synapses are surrounded by a layer of extracellular matrix (ECM), which is instrumental for their development and maintenance. ECM composition is dynamically controlled by proteases, but how the precise composition of the ECM affects synaptic morphology is largely unknown. Through an unbiased forward genetic screen, we found that Caenorhabditis elegans gon-1, a conserved extracellular ADAMTS protease, is required for maintaining proper synaptic morphology at the neuromuscular junction. In gon-1 mutants, once synapse formation is complete, motor neuron presynaptic varicosities develop into large bulbous protrusions that contain synaptic vesicles and active zone proteins. A concomitant overgrowth of postsynaptic muscle membrane is found in close apposition to presynaptic axonal protrusions. Mutations in the muscle-specific, actin-severing protein cofilin (unc-60) suppress the axon phenotype, suggesting that muscle outgrowth is necessary for presynaptic protrusions. gon-1 mutants can also be suppressed by loss of the ECM components collagen IV (EMB-9) and fibulin (FBL-1). We propose that GON-1 regulates a developmental switch out of an initial "pro-growth" phase during which muscle arms grow out and form synapses with motor neuron axons. We postulate that this switch involves degradation or reorganization of collagen IV (EMB-9), whereas FBL-1 opposes GON-1 by stabilizing EMB-9. Our results describe a mechanism for regulating synaptic ECM composition and reveal the importance of precise ECM composition for neuronal morphology and synapse integrity.


Mortality in persons with disability pension due to common mental disorders: A cohort study of Swedish construction workers.
Aims: This study investigated mortality in disability pensions due to common mental disorder, and variation over time after first receiving disability pension. Methods: Objectives were explored in 301,863 construction workers (97.2% men) recruited through healthcare examinations from 1971-1993. By linking with the Swedish National Insurance Agency registers, disability pensions until 2014 were identified. Common mental disorder was defined as disability pension diagnosis due to anxiety, stress-related disorders or moderate depression. Mortality was calculated in all-psychiatric diagnosis and diagnostic sub-groups, and compared to persons without disability pensions, using Poisson regression. Additional analyses were stratified by age at follow-up. Results: In total 6030 subjects received disability pensions based on psychiatric diagnoses, and 2624 constituted common mental disorder. Analyses in an all-psychiatric diagnosis displayed increased mortality risks in men (relative risk 3.6; 95% confidence interval 3.3-3.9) and women (relative risk 2.1; 95% confidence interval 1.6-2.6). Common mental disorder was associated with mortality, especially in men (relative risk 2.5; 95% confidence interval 2.2-2.8). Increased relative risks in alcohol and substance abuse were also observed. Results in analyses stratified by age at follow-up displayed persistent high relative risks for mortality in older ages (75-89 years) in men in all-psychiatric disability pensions diagnosis (relative risk 2.8; 95% confidence interval 2.1-3.7) and common mental disorder diagnosis (relative risk 2.6; 95% confidence interval 1.8-3.6), compared to men without disability pensions. Similar results were found in women, but few cases lowered the precision of estimates. Conclusions: This study shows that disability pension based on common mental disorders, often regarded as a 'lighter' psychiatric diagnosis, is a risk for early mortality in construction workers, even several years after first receiving disability pension.


Synthesis, X-ray crystal structures and biomimetic and anticancer activities of novel copper(II)benzoate complexes incorporating 2-(4'-thiazolyl)benzimidazole (thiabendazole), 2-(2-pyridyl)benzimidazole and 1,10-phenanthroline as chelating nitrogen donor ligands.
Cu(BZA)(2)(EtOH)(0.5) (1) was generated by the reaction of copper(II) hydroxide with benzoic acid (BZAH). [Cu(TBZH)(2)(BZA)](BZA).0.5TBZH.H(2)O (2) and [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)].1.66EtOH (3) were obtained when 1 reacted with Thiabendazole (TBZH) and 2-(2-pyridyl)benzimidazole (2-PyBZIMH), respectively. [Cu(BZA)(2)(phen)(H(2)O)] (4) was isolated from the reaction of benzoic acid and 1,10-phenanthroline (phen) with copper(II)acetate dihydrate. Molecular structures of 2, 3 and 4 were determined crystallographically. 2 and 3 are hydrogen bonded dimers and trimers, respectively. The copper centres in complexes 2 and 3 are bis-chelate derivatives that have N(4)O ligation and their geometry is very similar being approximately square-pyramidal. However whereas in complex 2 both TBZH ligands are neutral in 3 one of the 2-PyBZIMH chelators is deprotonated on each copper. The structural results for 4 represent a re-examination of this crystallographically known compound for which no hydrogen atom coordinates have been previously reported. It crystallises as a hydrogen bonded dimmer and is a mono-chelate of phen with each copper centre possessing N(2)O(3) ligation and square pyramidal geometry. The catalase and superoxide dismutase (SOD) activities of the four complexes along with those of the known phenanthroline complexes [Cu(mal)(phen)(2)] and [Cu(phendione)(3)](ClO(4))(2) (malH(2)=malonic acid and phendione=1,10-phenanthroline-5,6-dione) were investigated. Complexes 1-4, the metal free ligands and a simple copper(II) salt were assessed for their cancer chemotherapeutic potential against the hepatocellular carcinoma (Hep-G(2)) and kidney adenocarcinoma (A-498) cell lines. TBZH, 2-PyBZIMH and benzoic acid when uncoordinated to a metal centre offer poor chemotherapeutic potential. copper(II) benzoate is significantly more active than the free acid. The bis-chelate derivatives [Cu(TBZH)(2)(BZA)](BZA).0.5TBZH.H(2)O (2) and [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)].1.66EtOH (3) elicit a significant cytotoxic response to the cancer cell lines tested. Replacing TBZH and 2-PyBZIMH with phen to give [Cu(BZA)(2)(phen)(H(2)O)] (4) does not significantly increase the anti-cancer activity.


Subsequent neoplasms in 5-year survivors of childhood cancer: the Childhood Cancer Survivor Study.
The occurrence of subsequent neoplasms has direct impact on the quantity and quality of life in cancer survivors. We have expanded our analysis of these events in the Childhood Cancer Survivor Study (CCSS) to better understand the occurrence of these events as the survivor population ages. The incidence of and risk for subsequent neoplasms occurring 5 years or more after the childhood cancer diagnosis were determined among 14,359 5-year survivors in the CCSS who were treated from 1970 through 1986 and who were at a median age of 30 years (range = 5-56 years) for this analysis. At 30 years after childhood cancer diagnosis, we calculated cumulative incidence at 30 years of subsequent neoplasms and calculated standardized incidence ratios (SIRs), excess absolute risks (EARs) for invasive second malignant neoplasms, and relative risks for subsequent neoplasms by use of multivariable Poisson regression. Among 14,359 5-year survivors, 1402 subsequently developed 2703 neoplasms. Cumulative incidence at 30 years after the childhood cancer diagnosis was 20.5% (95% confidence interval [CI] = 19.1% to 21.8%) for all subsequent neoplasms, 7.9% (95% CI = 7.2% to 8.5%) for second malignant neoplasms (excluding nonmelanoma skin cancer), 9.1% (95% CI = 8.1% to 10.1%) for nonmelanoma skin cancer, and 3.1% (95% CI = 2.5% to 3.8%) for meningioma. Excess risk was evident for all primary diagnoses (EAR = 2.6 per 1000 person-years, 95% CI = 2.4 to 2.9 per 1000 person-years; SIR = 6.0, 95% CI = 5.5 to 6.4), with the highest being for Hodgkin lymphoma (SIR = 8.7, 95% CI = 7.7 to 9.8) and Ewing sarcoma (SIR = 8.5, 95% CI = 6.2 to 11.7). In the Poisson multivariable analysis, female sex, older age at diagnosis, earlier treatment era, diagnosis of Hodgkin lymphoma, and treatment with radiation therapy were associated with increased risk of subsequent neoplasm. As childhood cancer survivors progress through adulthood, risk of subsequent neoplasms increases. Patients surviving Hodgkin lymphoma are at greatest risk. There is no evidence of risk reduction with increasing duration of follow-up.


Diversity of bacterial communities along a petroleum contamination gradient in desert soils.
Microbial communities in oil-polluted desert soils have been rarely studied compared to their counterparts from freshwater and marine environments. We investigated bacterial diversity and changes therein in five desert soils exposed to different levels of oil pollution. Automated rRNA intergenic spacer (ARISA) analysis profiles showed that the bacterial communities of the five soils were profoundly different (analysis of similarities (ANOSIM), R = 0.45, P < 0.0001) and shared less than 20 % of their operational taxonomic units (OTUs). OTU richness was relatively higher in the soils with the higher oil pollution levels. Multivariate analyses of ARISA profiles revealed that the microbial communities in the S soil, which contains the highest level of contamination, were different from the other soils and formed a completely separate cluster. A total of 16,657 ribosomal sequences were obtained, with 42-89 % of these sequences belonging to the phylum Proteobacteria. While sequences belonging to Betaproteobacteria, Gammaproteobacteria, Bacilli, and Actinobacteria were encountered in all soils, sequences belonging to anaerobic bacteria from the classes Deltaproteobacteria, Clostridia, and Anaerolineae were only detected in the S soil. Sequences belonging to the genus Terriglobus of the class Acidobacteria were only detected in the B3 soil with the lowest level of contamination. Redundancy analysis (RDA) showed that oil contamination level was the most determinant factor that explained variations in the microbial communities. We conclude that the exposure to different levels of oil contamination exerts a strong selective pressure on bacterial communities and that desert soils are rich in aerobic and anaerobic bacteria that could potentially contribute to the degradation of hydrocarbons.


The Plasmodium falciparum pseudoprotease SERA5 regulates the kinetics and efficiency of malaria parasite egress from host erythrocytes.
Egress of the malaria parasite Plasmodium falciparum from its host red blood cell is a rapid, highly regulated event that is essential for maintenance and completion of the parasite life cycle. Egress is protease-dependent and is temporally associated with extensive proteolytic modification of parasite proteins, including a family of papain-like proteins called SERA that are expressed in the parasite parasitophorous vacuole. Previous work has shown that the most abundant SERA, SERA5, plays an important but non-enzymatic role in asexual blood stages. SERA5 is extensively proteolytically processed by a parasite serine protease called SUB1 as well as an unidentified cysteine protease just prior to egress. However, neither the function of SERA5 nor the role of its processing is known. Here we show that conditional disruption of the SERA5 gene, or of both the SERA5 and related SERA4 genes simultaneously, results in a dramatic egress and replication defect characterised by premature host cell rupture and the failure of daughter merozoites to efficiently disseminate, instead being transiently retained within residual bounding membranes. SERA5 is not required for poration (permeabilization) or vesiculation of the host cell membrane at egress, but the premature rupture phenotype requires the activity of a parasite or host cell cysteine protease. Complementation of SERA5 null parasites by ectopic expression of wild-type SERA5 reversed the egress defect, whereas expression of a SERA5 mutant refractory to processing failed to rescue the phenotype. Our findings implicate SERA5 as an important regulator of the kinetics and efficiency of egress and suggest that proteolytic modification is required for SERA5 function. In addition, our study reveals that efficient egress requires tight control of the timing of membrane rupture.


Lifestyle modifications to prevent and control hypertension. 5. Recommendations on dietary salt. Canadian Hypertension Society, Canadian Coalition for High Blood Pressure Prevention and Control, Laboratory Centre for Disease Control at Health Canada, Heart and Stroke Foundation of Canada.
To provide updated, evidence-based recommendations concerning the effects of dietary salt intake on the prevention and control of hypertension in adults (except pregnant women). The guidelines are intended for use in clinical practice and public education campaigns. Restriction of dietary salt intake may be an alternative to antihypertensive medications or may supplement such medications. Other options include other nonpharmacologic treatments for hypertension and no treatment. The health outcomes considered were changes in blood pressure and in morbidity and mortality rates. Because of insufficient evidence, no economic outcomes were considered. A MEDLINE search was conducted for the period 1966-1996 using the terms hypertension, blood pressure, vascular resistance, sodium chloride, sodium, diet, sodium or sodium chloride dietary, sodium restricted/reducing diet, clinical trials, controlled clinical trial, randomized controlled trial and random allocation. Both trials and review articles were obtained, and other relevant evidence was obtained from the reference lists of the articles identified, from the personal files of the authors and through contacts with experts. The articles were reviewed, classified according to study design and graded according to level of evidence. In addition, a systematic review of all published randomized controlled trials relating to dietary salt intake and hypertension was conducted. A high value was placed on the avoidance of cardiovascular morbidity and premature death caused by untreated hypertension. For normotensive people, a marked change in sodium intake is required to achieve a modest reduction in blood pressure (there is a decrease of 1 mm Hg in systolic blood pressure for every 100 mmol decrease in daily sodium intake). For hypertensive patients, the effects of dietary salt restriction are most pronounced if age is greater than 44 years. A decrease of 6.3 mm Hg in systolic blood pressure and 2.2 mm Hg in diastolic blood pressure per 100 mmol decrease in daily sodium intake was observed in people of this age group. For hypertensive patients 44 years of age and younger, the decreases were 2.4 mm Hg for systolic blood pressure and negligible for diastolic blood pressure. A diet in which salt is moderately restricted appears not to be associated with health risks. (1) Restriction of salt intake for the normotensive population is not recommended at present, because of insufficient evidence demonstrating that this would lead to a reduced incidence of hypertension. (2) To avoid excessive intake of salt, people should be counselled to choose foods low in salt (e.g., fresh fruits and vegetables), to avoid foods high in salt (e.g., pre-prepared foods), to refrain from adding salt at the table and minimize the amount of salt used in cooking, and to increase awareness of the salt content of food choices in restaurants. (3) For hypertensive patients, particularly those over the age of 44 years, it is recommended that the intake of dietary sodium be moderately restricted, to a target range of 90-130 mmol per day (which corresponds to 3-7 g of salt per day). (4) The salt consumption of hypertensive patients should be determined by interview. These recommendations were reviewed by all of the sponsoring organizations and by participants in a satellite symposium of the fourth International Conference on Preventive Cardiology. They have not been clinically tested. The Canadian Hypertension Society, the Canadian Coalition for High Blood Pressure Prevention and Control, the Laboratory Centre for Disease Control at Health Canada, and the Heart and Stroke Foundation of Canada.


A Population-Based Study of Genes Previously Implicated in Breast Cancer.
Population-based estimates of the risk of breast cancer associated with germline pathogenic variants in cancer-predisposition genes are critically needed for risk assessment and management in women with inherited pathogenic variants. In a population-based case-control study, we performed sequencing using a custom multigene amplicon-based panel to identify germline pathogenic variants in 28 cancer-predisposition genes among 32,247 women with breast cancer (case patients) and 32,544 unaffected women (controls) from population-based studies in the Cancer Risk Estimates Related to Susceptibility (CARRIERS) consortium. Associations between pathogenic variants in each gene and the risk of breast cancer were assessed. Pathogenic variants in 12 established breast cancer-predisposition genes were detected in 5.03% of case patients and in 1.63% of controls. Pathogenic variants in BRCA1 and BRCA2 were associated with a high risk of breast cancer, with odds ratios of 7.62 (95% confidence interval [CI], 5.33 to 11.27) and 5.23 (95% CI, 4.09 to 6.77), respectively. Pathogenic variants in PALB2 were associated with a moderate risk (odds ratio, 3.83; 95% CI, 2.68 to 5.63). Pathogenic variants in BARD1, RAD51C, and RAD51D were associated with increased risks of estrogen receptor-negative breast cancer and triple-negative breast cancer, whereas pathogenic variants in ATM, CDH1, and CHEK2 were associated with an increased risk of estrogen receptor-positive breast cancer. Pathogenic variants in 16 candidate breast cancer-predisposition genes, including the c.657_661del5 founder pathogenic variant in NBN, were not associated with an increased risk of breast cancer. This study provides estimates of the prevalence and risk of breast cancer associated with pathogenic variants in known breast cancer-predisposition genes in the U.S. population. These estimates can inform cancer testing and screening and improve clinical management strategies for women in the general population with inherited pathogenic variants in these genes. (Funded by the National Institutes of Health and the Breast Cancer Research Foundation.).


Genome scan meta-analysis of schizophrenia and bipolar disorder, part III: Bipolar disorder.
Genome scans of bipolar disorder (BPD) have not produced consistent evidence for linkage. The rank-based genome scan meta-analysis (GSMA) method was applied to 18 BPD genome scan data sets in an effort to identify regions with significant support for linkage in the combined data. The two primary analyses considered available linkage data for "very narrow" (i.e., BP-I and schizoaffective disorder-BP) and "narrow" (i.e., adding BP-II disorder) disease models, with the ranks weighted for sample size. A "broad" model (i.e., adding recurrent major depression) and unweighted analyses were also performed. No region achieved genomewide statistical significance by several simulation-based criteria. The most significant P values (<.01) were observed on chromosomes 9p22.3-21.1 (very narrow), 10q11.21-22.1 (very narrow), and 14q24.1-32.12 (narrow). Nominally significant P values were observed in adjacent bins on chromosomes 9p and 18p-q, across all three disease models on chromosomes 14q and 18p-q, and across two models on chromosome 8q. Relatively few BPD pedigrees have been studied under narrow disease models relative to the schizophrenia GSMA data set, which produced more significant results. There was no overlap of the highest-ranked regions for the two disorders. The present results for the very narrow model are promising but suggest that more and larger data sets are needed. Alternatively, linkage might be detected in certain populations or subsets of pedigrees. The narrow and broad data sets had considerable power, according to simulation studies, but did not produce more highly significant evidence for linkage. We note that meta-analysis can sometimes provide support for linkage but cannot disprove linkage in any candidate region.


Modulating effect of flavonoids and their derivatives on sarcoplasmic reticulum Ca2+-ATPase oxidized by hypochloric acid and peroxynitrite.
Effect of rutin and its lipophilic derivatives on Ca2+-ATPase of sarcoplasmic reticulum (SERCA) oxidized by hypochloric acid and peroxynitrite was investigated to examine the role of flavonoids in SERCA activity modulation. Ca2+-ATPase activity was measured spectrophotometrically at 37 degrees C using NADH-coupled enzyme pyruvate kinase/lactate dehydrogenase assay. SERCA was oxidized by HOCl (3 min) or ONOO- (30 s) after previous treatment with flavonoids (2 min) at 37 degrees C. Lipophilic rutin derivatives were prepared by lipase-catalyzed esterification of flavonoids with fatty acids. Both hypochloric acid (HOCl) and peroxynitrite (ONOO-) decreased ATPase activity concentration-dependently with IC50 of 50+/-10 micromol/l and 150+/-15 micromol/l, respectively. Rutin was found to have a protective effect on SERCA activity in both oxidation systems in the concentration range 5 - 250 micromol/l. Lipophilic rutin derivatives (rutin oleate, rutin linoleate, rutin linolenate) exerted inhibitory effect on ATPase activity both in the presence and absence of oxidants. The results suggest that selective lipophilization of the flavonoid skeleton may represent a useful tool for SERCA activity modulation.


An investigation of porphyrinuria in Australian children with autism.
Two recent studies, from France (Nataf et al., 2006) and the United States (Geier & Geier, 2007), identified atypical urinary porphyrin profiles in children with an autism spectrum disorder (ASD). These profiles serve as an indirect measure of environmental toxicity generally, and mercury (Hg) toxicity specifically, with the latter being a variable proposed as a causal mechanism of ASD (Bernard et al., 2001; Mutter et al., 2005). To examine whether this phenomenon occurred in a sample of Australian children with ASD, an analysis of urinary porphyrin profiles was conducted. A consistent trend in abnormal porphyrin levels was evidenced when data was compared with those previously reported in the literature. The results are suggestive of environmental toxic exposure impairing heme synthesis. Three independent studies from three continents have now demonstrated that porphyrinuria is concomitant with ASD, and that Hg may be a likely xenobiotic to produce porphyrin profiles of this nature.


Multiple types of motives don't multiply the motivation of West Point cadets.
Although people often assume that multiple motives for doing something will be more powerful and effective than a single motive, research suggests that different types of motives for the same action sometimes compete. More specifically, research suggests that instrumental motives, which are extrinsic to the activities at hand, can weaken internal motives, which are intrinsic to the activities at hand. We tested whether holding both instrumental and internal motives yields negative outcomes in a field context in which various motives occur naturally and long-term educational and career outcomes are at stake. We assessed the impact of the motives of over 10,000 West Point cadets over the period of a decade on whether they would become commissioned officers, extend their officer service beyond the minimum required period, and be selected for early career promotions. For each outcome, motivation internal to military service itself predicted positive outcomes; a relationship that was negatively affected when instrumental motives were also in evidence. These results suggest that holding multiple motives damages persistence and performance in educational and occupational contexts over long periods of time.


Inhibitory effect of yogurt on aberrant crypt foci formation in the rat colon and colorectal tumorigenesis in RasH2 mice.
The inhibitory effects of yogurt consisting of milk fermented by Lactobacillus delbrueckii subsp. bulgaricus strain 2038 and Streptococcus salivarius subsp. thermophilus strain 1131 on formation of colonic aberrant crypt foci (ACF) in rats and also on development of colorectal tumors in transgenic mice harboring human prototype c-Ha-ras genes (rasH2 mice) were examined. F344 rats and rasH2 mice were fed commercial diet containing freeze-dried yogurt or starter medium (non-fermented milk). Rats were inoculated orally with heterocyclic amine 2-amino-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP) for two weeks. The rats were necropsied 14 days after the PhIP treatment, and ACF in the colon and rectum were counted. RasH2 mice were injected with 1,2-dimethylhydrazine dihydrochloride (DMH) for 20 weeks. Three weeks after the last injection of DMH, rasH2 mice were necropsied to determine the number and the size of colorectal tumors. Yogurt supplementation in diet significantly reduced the number of ACF and aberrant crypts (ACs) in rats fed control diet (P<0.01), but not in rats fed non-fermented milk diet. On the other hand, rasH2 mice receiving the yogurt-supplemented diet had significantly reduced numbers of tumors induced by DMH compared with those fed the non-fermented milk-supplemented diet (P<0.05). These results demonstrate that the yogurt used in this study appears to have tumor-suppressing properties, and rasH2 mice are a useful model for the evaluation of antitumor activities of foods.


Effects of ethanol on antioxidant capacity in isolated rat hepatocytes.
To investigate dose-response and time-course of the effects of ethanol on the cell viability and antioxidant capacity in isolated rat hepatocytes. Hepatocytes were isolated from male adult Wistar rats and seeded into 100-mm dishes. Hepatocytes were treated with ethanol at concentrations between 0 (C), 10 (E10), 50 (E50), and 100 (E100) mmol/L (dose response) for 12, 24, and 36 h (time course). Then, lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) concentration, glutathione (GSH) level, and activities of glutathione peroxidase (GPX), glutathione reductase (GRD), superoxide dismutase (SOD), and catalase (CAT) were measured. Our data revealed that LDH leakage was significantly increased by about 30% in group E100 over those in groups C and E10 at 24 and 36 h, The MDA concentration in groups C, E10 and E50 were significantly lower than that in group E100 at 36 h. Furthermore, the concentration of MDA in group E100 at 36 h was significantly higher by 4.5- and 1.7-fold, respectively, than that at 12 and 24 h. On the other hand, the GSH level in group E100 at 24 and 36 h was significantly decreased, by 32% and 28%, respectively, compared to that at 12 h. The activities of GRD and CAT in group E100 at 36 h were significantly less than those in groups C and E10. However, The GPX and SOD activities showed no significant change in each group. These results suggest that long-time incubation with higher concentration of ethanol (100 mmol/L) decreased the cell viability by means of reducing GRD and CAT activities and increasing lipid peroxidation.


Male sex pheromone release and female mate choice in a butterfly.
In butterflies female mate choice is influenced by both visual and olfactory cues, the latter of which are important at close range. Males of the green-veined butterfly, Pieris napi, are known to release citral (mixture of geranial and neral, 1:1), but its role(s) and conditions of release are not known. Here, we show that male P. napi release citral when interacting with conspecific males, conspecific females, heterospecific males and also when alone. The amount of citral released correlated strongly with male flight activity, which explained more than 70% of the variation. This suggests that males do not exercise control over turning release on or off, but rather that citral is emitted as a passive physical process during flight. Electroantennogram experiments showed that female antennal response was ten times more sensitive to citral than male response. Females expressed acceptance behavior when exposed to models made with freshly excised male wings or those treated with citral following chemical extraction, but not to ones with extracted wings only. Hence, these behavioral and electrophysiological tests provide strong evidence that citral is a signal from the male directed to the female during courtship, and that it functions as a male sex pheromone.


Peptide inhibitors disrupt the serotonin 5-HT2C receptor interaction with phosphatase and tensin homolog to allosterically modulate cellular signaling and behavior.
Serotonin (5-hydroxytryptamine; 5-HT) signaling through the 5-HT(2C) receptor (5-HT(2C)R) is essential in normal physiology, whereas aberrant 5-HT(2C)R function is thought to contribute to the pathogenesis of multiple neural disorders. The 5-HT(2C)R interacts with specific protein partners, but the impact of such interactions on 5-HT(2C)R function is poorly understood. Here, we report convergent cellular and behavioral data that the interaction between the 5-HT(2C)R and protein phosphatase and tensin homolog (PTEN) serves as a regulatory mechanism to control 5-HT(2C)R-mediated biology but not that of the closely homologous 5-HT(2A)R. A peptide derived from the third intracellular loop of the human 5-HT(2C)R [3L4F (third loop, fourth fragment)] disrupted the association, allosterically augmented 5-HT(2C)R-mediated signaling in live cells, and acted as a positive allosteric modulator in rats in vivo. We identified the critical residues within an 8 aa fragment of the 3L4F peptide that maintained efficacy (within the picomolar range) in live cells similar to that of the 3L4F peptide. Last, molecular modeling identified key structural features and potential interaction sites of the active 3L4F peptides against PTEN. These compelling data demonstrate the specificity and importance of this protein assembly in cellular events and behaviors mediated by 5-HT(2C)R signaling and provide a chemical guidepost to the future development of drug-like peptide or small-molecule inhibitors as neuroprobes to study 5-HT(2C)R allostery and therapeutics for 5-HT(2C)R-mediated disorders.


Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development.
To address the function of bone morphogenetic protein-2 (BMP2) in mammalian development, mice with a targeted deletion of the Bmp2 mature region were generated using embryonic stem cell technology. This mutation caused embryonic lethality when homozygous. Mutant embryos failed to close the proamniotic canal, which caused the malformation of the amnion/chorion. BMP2-deficient embryos also exhibited a defect in cardiac development, manifested by the abnormal development of the heart in the exocoelomic cavity. These defects are consistent with the expression of Bmp2 in the extraembryonic mesoderm cells and promyocardium. Thus BMP2 is a critical factor for both extraembryonic and embryonic development.


Potentiation of norepinephrine secretion by angiotensin II in the isolate rabbit iris-ciliary body.
The prejunctional effects of angiotensin II (AII) on stimulation-evoked secretion of 3H-norepinephrine (3H-NE) were investigated by in vitro methods in isolated, superfused rabbit iris-ciliary body preparations. AII (0.1-10 nM) concentration-dependently enhanced the field- stimulated release of 3H-NE (EC50 = 0.1 nM), nearly doubling evoked neurotransmitter release with no apparent effect on spontaneous 3H-NE efflux. The response to 1 nM AII was abolished by the selective AII receptor antagonist saralasin [( Sar1,Val5, Ala8]-angiotensin II; 500 nM), which alone did not modify 3H-NE overflow. AII-mediated effects on neurosecretion were partially additive to those of forskolin and were not potentiated by phosphodiesterase inhibition, suggesting that AII utilizes a mechanism other than increased cAMP synthesis to facilitate neurotransmitter release. AII also strongly enhanced calcium ionophore (A23187)-induced 3H-NE release in iris-ciliary body segments, indicating that AII can modulate calcium-dependent exocytosis at step(s) distal to calcium influx. These results demonstrate that sympathetic nerves in the rabbit eye contain prejunctional, facilitatory AII receptors, and support the possible involvement of the renin-angiotensin system in regulation of ocular sympathetic neurotransmission in vivo.


Fighting Off Wound Pathogens in Horses with Honeybee Lactic Acid Bacteria.
In the global perspective of antibiotic resistance, it is urgent to find potent topical antibiotics for the use in human and animal infection. Healing of equine wounds, particularly in the limbs, is difficult due to hydrostatic factors and exposure to environmental contaminants, which can lead to heavy bio-burden/biofilm formation and sometimes to infection. Therefore, antibiotics are often prescribed. Recent studies have shown that honeybee-specific lactic acid bacteria (LAB), involved in honey production, and inhibit human wound pathogens. The aim of this pilot study was to investigate the effects on the healing of hard-to-heal equine wounds after treatment with these LAB symbionts viable in a heather honey formulation. For this, we included ten horses with wound duration of >1 year, investigated the wound microbiota, and treated wounds with the novel honeybee LAB formulation. We identified the microbiota using MALDI-TOF mass spectrometry and DNA sequencing. In addition, the antimicrobial properties of the honeybee LAB formulation were tested against all wound isolates in vitro. Our results indicate a diverse wound microbiota including fifty-three bacterial species that showed 90 % colonization by at least one species of Staphylococcus. Treatment with the formulation promoted wound healing in all cases already after the first application and the wounds were either completely healed (n = 3) in less than 20 days or healing was in progress. Furthermore, the honeybee LAB formulation inhibited all pathogens when tested in vitro. Consequently, this new treatment option presents as a powerful candidate for the topical treatment of hard-to-heal wounds in horses.


High pressure mass spectrometry of volatile organic compounds with ambient air buffer gas.
There are many chemical measurement scenarios that would benefit from hand portable mass spectrometry tools including forensics, environmental monitoring, and safety and security. High pressure mass spectrometry (HPMS) facilitates miniaturization by significantly reducing vacuum system requirements. Previous work demonstrated HPMS using helium buffer gas, but HPMS conducted using ambient air would further reduce the size and weight of a portable instrument while also reducing logistical demands by eliminating the need for a helium supply. Mass spectrometry was performed at pressures exceeding 1 Torr with ambient air as the buffer gas. A glow discharge electron ionization source and a miniature cylindrical ion trap mass analyzer with a radius of 0.5 mm were used. Mass analysis was possible at these pressures with increased radiofrequency (RF) drive frequencies (10 MHz) compared with commercial ion traps (~1 MHz). A differentially pumped chamber was used so that mass spectrometry could be performed at high pressures and detection performed at low pressures with an electron multiplier. HPMS with air buffer gas was demonstrated using a suite of volatile organic compounds (VOCs). The glow discharge ionization source was optimized for operation using air. Mass spectral peak widths increased a factor of 8 compared with helium, as expected, but useful chemical information was still acquired. A mixture of VOCs was detected with ambient air as the buffer gas, showing that valuable mass information can be gained using HPMS without the requirement of an onboard buffer gas source. HPMS significantly reduces the pumping requirements required for miniature mass spectrometers and the use of ambient air buffer gas further reduces size, weight, and logistics requirements. Mass analysis at high pressures of ambient air is another important step for the development of hand portable mass spectrometers. Copyright © 2016 John Wiley & Sons, Ltd.


Improving understanding of clinical trial procedures among low literacy populations: an intervention within a microbicide trial in Malawi.
The intervention reported in this paper was a follow up to an empirical study conducted in Malawi with the aim of assessing trial participants' understanding of randomisation, double-blinding and placebo use. In the empirical study, the majority of respondents (61.1%; n=124) obtained low scores (lower than 75%) on understanding of all three concepts under study. Based on these findings, an intervention based on a narrative which included all three concepts and their personal implications was designed. The narrative used daily examples from the field of Agriculture because Malawi has an agro-based economy. The intervention was tested using a sample of 36 women who had been identified as low scorers during the empirical study. The 36 low scorers were randomly assigned to control (n=18) and intervention arms (n=18). The control arm went through a session in which they were provided with standard informed consent information for the microbicide trial. The intervention arm went through a session in which they were provided with a narrative in ChiChewa, the local language, with the assistance of a power point presentation which included pictures as well as discussions on justification and personal implications of the concepts under study. The findings on the efficacy of the intervention suggest that the 3 scientific concepts and their personal implications can be understood by low literacy populations using simple language and everyday local examples. The findings also suggest that the intervention positively impacted on understanding of trial procedures under study, as 13 of the 18 women in the intervention arm, obtained high scores (above 75%) during the post intervention assessment and none of the 18 in the control arm obtained a high score. Using Fischer's exact test, it was confirmed that the effect of the intervention on understanding of the three procedures was statistically significant (p=0.0001). Potential trial participants can be assisted to understand key clinical trial procedures, their justification and personal implications by using innovative tailored local narratives.


Slowing down to stay out of trouble in the operating room: remaining attentive in automaticity.
Automaticity is integral to expert performance, but experts must be able to transition from an automatic mode into a more effortful state when required. In this study, the authors identified and characterized the manifestations of the phenomenon of "slowing down when you should" to stay out of trouble in operative practice. The authors interviewed 28 surgeons (60-minute, semistructured format) from various specialties at four academic medical centers and observed 5 hepatopancreatobiliary surgeons in the operating room (29 cases, 147 hours) during 2007-2009. Using a grounded theory qualitative methodology, they conducted a thematic analysis of transcripts and field notes in an iterative manner. Data collection continued until saturation. They adopted a reflexive approach throughout. Surgeons described and the authors observed four phenomenological manifestations of the transition to a more effortful state. In the most extreme manifestation, "stopping," surgeons actually stopped the procedure, whereas in the most subtle manifestation, "fine-tuning," surgeons were able to continue the procedure and focus on minor events simultaneously. A separate phenomenon of "drifting" represented surgeons' failure to transition out of the automatic mode when appropriate, resulting in surgical errors or near misses. The manifestations of the slowing down phenomenon represent acts of cognitive refocusing during the potentially more-critical moments of operative practice. Further, the authors challenge the conception of automaticity as effortless, arguing that automatic behavior can be attentive (fine-tuning) as well as inattentive (drifting).


Clinical Effectiveness of a Combination of Black Elder Berries, Violet Herb, and Calendula Flowers in Chronic Obstructive Pulmonary Disease: The Results of a Double-Blinded Placebo-Controlled Study.
Chronic obstructive pulmonary disease (COPD) is a multifactorial disease, in which systemic inflammation plays a key role. This 6-month randomized double-blinded placebo-controlled study evaluates the possible effect of natural preparation Inflaminat on clinical symptoms of COPD, indicators of respiratory function, and exacerbation frequency in 60 patients with moderate severity of COPD. Inflaminat is a combination of natural ingredients black elder (Sambucus nigra L.) berries, violet (Viola tricolor L.) herb, and calendula (Calendula officinalis L.) flowers. The preparation has been previously demonstrated to possess anticytokine and anti-inflammatory effects in experimental studies. In present study, COPD dynamics were evaluated by means of BCSS (Breathlessness, Cough, and Sputum Scale) and spirometry tests. It was shown that 6-months Inflaminat administration led to significant decrease of BCSS points from 3.0 ± 0.6 to 1.9 ± 0.7, (p = 0.002) as well as significant increase of FEV1 from 66 ± 18% to 73 ± 17%, (p = 0.042); there were no beneficial dynamics in placebo group. Side effects associated with preparation administration were not identified. The results of the study suggest that Inflaminat may be employed in treatment of patients with moderate severity of COPD, since it has a positive effect on COPD symptoms according BCSS and indicators of respiratory function FEV1.


PAP1 signaling involves MAPK signal transduction.
Pancreatitis-associated protein 1 (PAP1) belongs to the Reg family of secretory proteins. Several important biological roles have been attributed to PAP1 but the signaling pathways activated by this protein remain only partially understood. Here, we describe the intracellular pathways triggered by PAP1 in a pancreatic acinar cell line. Taking advantage of the fact that PAP1 induces its own transcription, we performed ChIP assays to analyze the recruitment of transcriptional factors on its promoter. Our results show that PAP1 increased the transactivation activity of pap1 and the binding on its promoter of the nuclear factors C/EBPbeta, P-CREB, P-ELK1, EGR1, STAT3, and ETS2, which are downstream targets of MAPK signaling. p44/42, p38, and JNK MAPKs activity increased after PAP1 treatment. In addition, pharmacological inhibition of these kinases markedly inhibited the induction of pap1 mRNA. Taken together, these results indicated that the mechanism of PAP1 action involves the activation of the MAPK superfamily.


Dexamethasone and DMSO in experimental transorbital cerebral infarction.
Twenty rhesus monkeys were used to evaluate the effects of dexamethasone and dimethyl sulfoxide (DMSO) following experimental occlusion of the middle cerebral artery (MCA) for 17 hours. Animals were initially treated after four hours while the MCA remained occluded and a series of tests was used to evaluate the neurological and cerebral status of each monkey for seven days. The results show that the gross, microscopic and angiographical picture of dexamethasone and no-treatment controls was practically identical. In contrast, DMSO-treated monkeys showed significant protection from the severe neurological deficits seen in the other groups. It is concluded that DMSO has a positive effect in reducing the neurological deficits seen in this model and may be potentially useful in clinical emoblic stroke.


Experimental evaluation of local wave speed in the presence of reflected waves.
Wave speed (also called pulse wave velocity) is the speed by which disturbance travels along the medium and it depends on the mechanical and geometrical properties of the vessel and on the density of the blood. Wave speed is a parameter of clinical relevance because it is an indicator of arterial stiffness and cardiovascular diseases. The aim of this work is to compare different methods for the determination of local wave speed in bench experiments and investigate their relative accuracy when reflections are present. Pressure (P), flow (Q) and diameter (D) were measured along a flexible tube far and close to three positive and three negative reflection sites. Wave speed was calculated using PU-loop, (lnD)U-loop, QA-loop, D(2)P-loop, sum of squares and characteristic impedance methods. Results were compared to the foot-to-foot method. We found that far from the reflections almost all methods give uniform results. Close to positive reflections the methods that rely on P and Q (or U) overestimate the wave speed value, while techniques based on D (or A) and Q (or U) underestimate it. On the contrary, close to negative reflections the methods that rely on P and Q (or U) underestimate the wave speed value, while techniques based on D (or A) and Q (or U) overestimate it. The D(2)P-loop does not seem to be affected by positive or negative reflections. Most of the methods currently used to determine local wave speed are affected by reflections, but the (lnD)U-loop remains the easiest technique to use in the clinic.


Wait time management strategies for scheduled care: what makes them succeed?
To assess experts' perceptions of the contextual and local factors that promote or inhibit the implementation of waiting time management strategies (WTMS) in Canadian healthcare organizations. We conducted 16 semi-structured interviews and one focus group with individuals involved in WTMS at the federal, provincial or organizational level. The most frequently cited local factor was physicians' participation. Physicians' leadership made the greatest difference in bringing resistant physicians on board. To be effective, however, local leadership had to be supported by senior management. Alignment of financial incentives between the contextual and local levels was also frequently cited, and interviewees stressed the importance of tools used to design, monitor, evaluate and prioritize WTMS. Finding the right balance between supportive resources and tools and an effective management system is a tough challenge. But achieving this balance will help reconcile contradictions between top-down and bottom-up WTMS.


Common genetic determinants of vitamin D insufficiency: a genome-wide association study.
Vitamin D is crucial for maintenance of musculoskeletal health, and might also have a role in extraskeletal tissues. Determinants of circulating 25-hydroxyvitamin D concentrations include sun exposure and diet, but high heritability suggests that genetic factors could also play a part. We aimed to identify common genetic variants affecting vitamin D concentrations and risk of insufficiency. We undertook a genome-wide association study of 25-hydroxyvitamin D concentrations in 33 996 individuals of European descent from 15 cohorts. Five epidemiological cohorts were designated as discovery cohorts (n=16 125), five as in-silico replication cohorts (n=9367), and five as de-novo replication cohorts (n=8504). 25-hydroxyvitamin D concentrations were measured by radioimmunoassay, chemiluminescent assay, ELISA, or mass spectrometry. Vitamin D insufficiency was defined as concentrations lower than 75 nmol/L or 50 nmol/L. We combined results of genome-wide analyses across cohorts using Z-score-weighted meta-analysis. Genotype scores were constructed for confirmed variants. Variants at three loci reached genome-wide significance in discovery cohorts for association with 25-hydroxyvitamin D concentrations, and were confirmed in replication cohorts: 4p12 (overall p=1.9x10(-109) for rs2282679, in GC); 11q12 (p=2.1x10(-27) for rs12785878, near DHCR7); and 11p15 (p=3.3x10(-20) for rs10741657, near CYP2R1). Variants at an additional locus (20q13, CYP24A1) were genome-wide significant in the pooled sample (p=6.0x10(-10) for rs6013897). Participants with a genotype score (combining the three confirmed variants) in the highest quartile were at increased risk of having 25-hydroxyvitamin D concentrations lower than 75 nmol/L (OR 2.47, 95% CI 2.20-2.78, p=2.3x10(-48)) or lower than 50 nmol/L (1.92, 1.70-2.16, p=1.0x10(-26)) compared with those in the lowest quartile. Variants near genes involved in cholesterol synthesis, hydroxylation, and vitamin D transport affect vitamin D status. Genetic variation at these loci identifies individuals who have substantially raised risk of vitamin D insufficiency. Full funding sources listed at end of paper (see Acknowledgments).


Randomized trial of carboplatin versus radiotherapy for stage I seminoma: mature results on relapse and contralateral testis cancer rates in MRC TE19/EORTC 30982 study (ISRCTN27163214).
Initial results of a randomized trial comparing carboplatin with radiotherapy (RT) as adjuvant treatment for stage I seminoma found carboplatin had a noninferior relapse-free rate (RFR) and had reduced contralateral germ cell tumors (GCTs) in the short-term. Updated results with a median follow-up of 6.5 years are now reported. Random assignment was between RT and one infusion of carboplatin dosed at 7 × (glomerular filtration rate + 25) on the basis of EDTA (n = 357) and 90% of this dose if determined on the basis of creatinine clearance (n = 202). The trial was powered to exclude a doubling in RFRs assuming a 96-97% 2-year RFR after radiotherapy (hazard ratio [HR], approximately 2.0). Overall, 1,447 patients were randomly assigned in a 3-to-5 ratio (carboplatin, n = 573; RT, n = 904). RFRs at 5 years were 94.7% for carboplatin and 96.0% for RT (RT-C 90% CI, 0.7% to 3.5%; HR, 1.25; 90% CI, 0.83 to 1.89). One death as a result of seminoma (in RT arm) occurred. Patients receiving at least 99% of the 7 × AUC dose had a 5-year RFR of 96.1% (95% CI, 93.4% to 97.7%) compared with 92.6% (95% CI, 88.0% to 95.5%) in those who received lower doses (HR, 0.51; 95% CI, 0.24 to 1.07; P = .08). There was a clear reduction in the rate of contralateral GCTs (carboplatin, n = 2; RT, n = 15; HR, 0.22; 95% CI, 0.05 to 0.95; P = .03), and elevated pretreatment follicle-stimulating hormone (FSH) levels (> 12 IU/L) was a strong predictor (HR, 8.57; 95% CI, 1.82 to 40.38). These updated results confirm the noninferiority of single dose carboplatin (at 7 × AUC dose) versus RT in terms of RFR and establish a statistically significant reduction in the medium term of risk of second GCT produced by this treatment.


Spike-time reliability of layered neural oscillator networks.
We study the reliability of layered networks of coupled "type I" neural oscillators in response to fluctuating input signals. Reliability means that a signal elicits essentially identical responses upon repeated presentations, regardless of the network's initial condition. We study reliability on two distinct scales: neuronal reliability, which concerns the repeatability of spike times of individual neurons embedded within a network, and pooled-response reliability, which concerns the repeatability of total synaptic outputs from a subpopulation of the neurons in a network. We find that neuronal reliability depends strongly both on the overall architecture of a network, such as whether it is arranged into one or two layers, and on the strengths of the synaptic connections. Specifically, for the type of single-neuron dynamics and coupling considered, single-layer networks are found to be very reliable, while two-layer networks lose their reliability with the introduction of even a small amount of feedback. As expected, pooled responses for large enough populations become more reliable, even when individual neurons are not. We also study the effects of noise on reliability, and find that noise that affects all neurons similarly has much greater impact on reliability than noise that affects each neuron differently. Qualitative explanations are proposed for the phenomena observed.


Oh, the Places We'll Go: Patient-Reported Outcomes and Electronic Health Records.
The growing measurement of patient-reported outcomes (PROs) and adoption of electronic health records (EHRs) presents an unprecedented opportunity to improve health care for patients and populations. The integration of PROs into EHRs can promote patient-centered care and advance quality improvement initiatives, research, and population health. Despite these potential benefits, there are few best practices to help organizations achieve integration. To integrate PROs into EHRs, organizations should evaluate the advantages and disadvantages of various approaches within three themes: Planning, Selection, and Engagement. Planning considerations for integration include what strategy will be used, how the integrated system will be governed, ethical and legal issues, and how data from multiple EHRs can be pooled across organizations. Selection considerations involve identifying which patient population to target for PRO data collection on the basis of the intended use of the data in the health care system, and then choosing specific outcomes and their measures. Engagement considerations include how, where, and with what frequency patients will respond to PRO measures, how to display PRO data in EHRs, how clinical teams will act upon PRO data, and how to train, support and incent clinical teams and patients to incorporate PRO data into care. There is no most effective model that will work in all contexts. Organizations wishing to integrate PROs and EHRs should assemble the multidisciplinary expertise needed to evaluate the advantages and disadvantages of the various approaches for their particular context. We specifically recommend that organizations think carefully about stakeholder participation; design their system with data sharing in mind; develop a framework to aid in PRO selection; create guidelines to support PRO interpretation and action for patients and clinicians; and ensure patients have access to their own PRO data.


Comparing contents of outcome measures in cerebral palsy using the International Classification of Functioning (ICF-CY): a systematic review.
The International Classification of Functioning children and youth version (ICF-CY) provides a universal framework for defining and classifying functioning and disability in children worldwide. To facilitate the application of the ICF in practice, ICF based-tools like the "ICF Core Sets" are being developed. In the context of the development of the ICF-CY Core Sets for children with Cerebral Palsy (CP), the aims of this study were as follows: to identify and compare the content of outcome measures used in studies of children with CP using the ICF-CY coding system; and to describe the most frequently addressed areas of functioning in those studies. We searched multiple databases likely to capture studies involving children with CP from January 1998 to March 2012. We included all English language articles that studied children aged 2-18 years and described an interventional or observational study. Constructs of the outcome measures identified in studies were linked to the ICF-CY by two trained professionals. We found 231 articles that described 238 outcome measures. The outcome measures contained 2193 concepts that were linked to the ICF-CY and covered 161 independent ICF-CY categories. Out of the 161 categories, 53 (33.5%) were related to body functions, 75 (46%) were related to activities/participation, 26 (16.1%) were related to environmental factors, and 7 (4.3%) were related to body structures. This systematic review provides information about content of measures that may guide researchers and clinicians in their selection of an outcome measure for use in a study and/or clinical practice with children with CP.


Application of Thyroid Imaging Reporting and Data System in the Ultrasound Assessment of Thyroid Nodules According to Physician Experience.
The aim of this study was to investigate and compare the diagnostic performances of the Thyroid Imaging Reporting and Data System (TIRADS) in differentiating benign and malignant thyroid nodules according to the level of physician experience. From March to October 2013, 1102 patients with 1128 thyroid nodules who underwent initial ultrasound-guided fine needle aspiration were included in this study. Thyroid nodules were categorized according to TIRADS. Diagnostic performances of ultrasound were compared according to performer experience using the χ test or Fisher exact test. Of 1128 thyroid nodules, 281 were malignant, and 847 were benign. The risk of malignancy of each TIRADS category by the experienced and less experienced physicians were as follows: category 3 (0.9% vs 0%), category 4a (3.5% vs 1.3%), category 4b (7.3% vs 12.1%), category 4c (67.5% vs 44.9%), and category 5 (97.7% vs 76.5%). Sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were 99.1%, 35.9%, 52.5%, 35.5%, and 99.1%, respectively, for experienced physicians and 100%, 20.9%, 37.6%, 35.2%, and 100%, respectively, for less experienced physicians. Specificity, accuracy, and positive predictive value were statistically higher for experienced physicians than those for less experienced physicians (P < 0.001, 0.001, and 0.004). There was a significant difference in areas under the curve between the 2 groups (P < 0.001). In conclusion, the diagnostic performance of the stratification of malignancy risk according to TIRADS categories was comparable between the experienced and less experienced physician groups. The application of TIRADS is reproducible, and it was easy to predict the probability of thyroid malignancy in both the experienced and less experienced physician groups.


Oxygen transport by low and normal oxygen affinity hemoglobin vesicles in extreme hemodilution.
The oxygen transport capacity of phospholipid vesicles encapsulating purified Hb (HbV) produced with a Po(2) at which Hb is 50% saturated (P 50 ) of 8 (HbV(8)) and 29 mmHg (HbV(29)) was investigated in the hamster chamber window model by using microvascular measurements to determine oxygen delivery during extreme hemodilution. Two isovolemic hemodilution steps were performed with 5% recombinant albumin (rHSA) until Hct was 35% of baseline. Isovolemic exchange was continued using HbV suspended in rHSA solution to a total [Hb] of 5.7 g/dl in blood. P(50) was modified by coencapsulating pyridoxal 5'-phosphate. Final Hct was 11% for the HbV groups, with a plasma [Hb] of 2.1 +/- 0.1 g/dl after exchange with HbV(8) or HbV(29). A reference group was hemodiluted to Hct 11% with only rHSA. All groups showed stable blood pressure and heart rate. Arterial oxygen tensions were significantly higher than baseline for the HbV groups and the rHSA group and significantly lower for the HbV groups compared with the rHSA group. Blood pressure was significantly higher for the HbV(8) group compared with the HbV(29) group. Arteriolar and venular blood flows were significantly higher than baseline for the HbV groups. Microvascular oxygen delivery and extraction were similar for the HbV groups but lower for the rHSA group (P < 0.05). Venular and tissue Po(2) were statistically higher for the HbV(8) vs. the HbV(29) and rHSA groups (P < 0.05). Improved tissue Po(2) is obtained when red blood cells deliver oxygen in combination with a high- rather than low-affinity oxygen carrier.


A comparison of oral artesunate and artemether antimalarial bioactivities in acute falciparum malaria.
Artesunate and artemether are the two most widely used artemisinin derivatives in the treatment of uncomplicated Plasmodium falciparum malaria, but there is little information on their comparative pharmacokinetics. The aim of this study was to examine the relative oral antimalarial bioavailability and pharmacokinetics of the two derivatives. The pharmacokinetic properties of oral artesunate and artemether (4 mg kg(-1)) were compared in a randomized cross-over study of 14 adult patients in western Thailand with acute uncomplicated Plasmodium falciparum malaria. Antimalarial activity was compared using a previously validated, sensitive bioassay. Despite a 29% lower molar dose, oral artesunate administration resulted in significantly larger mean area under the plasma antimalarial activity time curve and median maximum plasma antimalarial activity than after oral artemether (P <or= 0.02). The mean (95% CI) oral antimalarial bioavailability of artemether, relative to oral artesunate, corrected for molar dose was 58 (40-76)%. The mean (95% CI) relative antimalarial bioavailability of artemether was lower on the first day of treatment, 31 (17-100)%, compared to the second day, 72 (44-118)% (P = 0.018). In vivo parasite clearance and time above the in vitro IC90 were similar for the two drugs, despite considerable differences in Cmax and AUC. The oral antimalarial bioavailability following artemether was significantly lower than that after artesunate. Artemether oral antimalarial bioavailability is reduced in acute malaria.


The Dictyostelium prestalk inducer DIF-1 directs phosphorylation of a bZIP transcription factor.
DIF-1, a chlorinated hexaphenone produced by developing Dictyostelium cells, induces prestalk differentiation. DimB is a bZIP transcription factor that accumulates in the nucleus upon exposure to DIF-1, where it directly activates transcription of DIF-responsive genes. The signaling steps upstream of DimB and downstream of DIF-1 are entirely unknown. Analysis by mass spectrometry shows that incubation with DIF-1 rapidly stimulates phosphorylation at several sites in DimB. We characterize the most highly responsive site, S590, which is located very close to the C terminus. A point mutation in this site, S590A, does not inhibit DimB nuclear accumulation in response to DIF. However, this seems likely to reflect functional redundancy with other sites; because a panel of chemical variants on the structure of DIF-1 show a correlation between their potencies as inducers of DimB nuclear accumulation and their potencies as inducers of phosphorylation at S590. Furthermore, the S590A mutant is fully active in mutant rescue of a dimB null strain, arguing against an alternative role in transcriptional activation of target genes. We conclude that i) DIF-1 directs phosphorylation at S590, ii) although it is not essential for nuclear accumulation in response to DIF-1 correlative evidence, based upon a panel of DIF-1 related molecules, suggests that this modification may play a redundant role in the process. iii) We also present evidence that the kinase activity, which phosphorylates S590, is non-nuclear and that this signalling pathway is, in part at least, independent of the DIF-regulated STATc activation pathway.


A human XRCC4-XLF complex bridges DNA.
DNA double-strand breaks pose a significant threat to cell survival and must be repaired. In higher eukaryotes, such damage is repaired efficiently by non-homologous end joining (NHEJ). Within this pathway, XRCC4 and XLF fulfill key roles required for end joining. Using DNA-binding and -bridging assays, combined with direct visualization, we present evidence for how XRCC4-XLF complexes robustly bridge DNA molecules. This unanticipated, DNA Ligase IV-independent bridging activity by XRCC4-XLF suggests an early role for this complex during end joining, in addition to its more well-established later functions. Mutational analysis of the XRCC4-XLF C-terminal tail regions further identifies specialized functions in complex formation and interaction with DNA and DNA Ligase IV. Based on these data and the crystal structure of an extended protein filament of XRCC4-XLF at 3.94 Å, a model for XRCC4-XLF complex function in NHEJ is presented.


A third measure-metastable state in the dynamics of spontaneous shape change in healthy human's white cells.
Human polymorphonuclear leucocytes, PMN, are highly motile cells with average 12-15 µm diameters and prominent, loboid nuclei. They are produced in the bone marrow, are essential for host defense, and are the most populous of white blood cell types. PMN also participate in acute and chronic inflammatory processes, in the regulation of the immune response, in angiogenesis, and interact with tumors. To accommodate these varied functions, their behavior is adaptive, but still definable in terms of a set of behavioral states. PMN morphodynamics have generally involved a non-equilibrium stationary, spheroid Idling state that transitions to an activated, ellipsoid translocating state in response to chemical signals. These two behavioral shape-states, spheroid and ellipsoid, are generally recognized as making up the vocabulary of a healthy PMN. A third, "random" state has occasionally been reported as associated with disease states. I have observed this third, Treadmilling state, in PMN from healthy subjects, the cells demonstrating metastable dynamical behaviors known to anticipate phase transitions in mathematical, physical, and biological systems. For this study, human PMN were microscopically imaged and analyzed as single living cells. I used a microscope with a novel high aperture, cardioid annular condenser with better than 100 nanometer resolution of simultaneous, mixed dark field and intrinsic fluorescent images to record shape changes in 189 living PMNs. Relative radial roundness, R(t), served as a computable order parameter. Comparison of R(t) series of 10 cells in the Idling and 10 in the Treadmilling state reveals the robustness of the "random" appearing Treadmilling state, and the emergence of behaviors observed in the neighborhood of global state transitions, including increased correlation length and variance (divergence), sudden jumps, mixed phases, bimodality, power spectral scaling and temporal slowing. Wavelet transformation of an R(t) series of an Idling to Treadmilling state change, demonstrated behaviors concomitant with the observed transition.


US Competitiveness in Synthetic Biology.
Synthetic biology is an emerging technical field that aims to make biology easier to engineer; the field has applications in strategically important sectors for the US economy. While the United States currently leads in synthetic biology R&D, other nations are heavily investing in order to boost their economies, which will inevitably diminish the US leadership position. This outcome is not entirely negative--additional investments will expand markets--but it is critical that the US government take steps to remain competitive: There are applications from which the US population and economy may benefit; there are specific applications with importance for national defense; and US technical leadership will ensure that US experts have a leading role in synthetic biology governance, regulation, and oversight. Measures to increase competitiveness in S&T generally are broadly applicable for synthetic biology and should be pursued. However, the US government will also need to take action on fundamental issues that will affect the field's development, such as countering anti-GMO (genetically modified organism) sentiments and anti-GMO legislation. The United States should maintain its regulatory approach so that it is the product that is regulated, not the method used to create a product. At the same time, the United States needs to ensure that the regulatory framework is updated so that synthetic biology products do not fall into regulatory gaps. Finally, the United States needs to pay close attention to how synthetic biology applications may be governed internationally, such as through the Nagoya Protocol of the Convention on Biological Diversity, so that beneficial applications may be realized.


Molecular hydrogen ameliorates lipopolysaccharide-induced acute lung injury in mice through reducing inflammation and apoptosis.
Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our and other studies have found that hydrogen gas (H₂) treatment can ameliorate the lung injury induced by sepsis, ventilator, hyperoxia, and ischemia-reperfusion. However, the molecular mechanisms by which H₂ ameliorates lung injury remain unclear. In the current study, we investigated whether H₂ or hydrogen-rich saline (HS) could exert protective effects in a mouse model of ALI induced by intratracheal administration of lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway-mediated inflammation and apoptosis. Two percent of H₂ was inhaled for 1 h beginning at 1 and 6 h after LPS administration, respectively. We found that LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology and histologic scores, wet-to-dry weight ratio, and oxygenation index (PaO₂/FIO₂), as well as total protein in the bronchoalveolar lavage fluid (BALF), which was attenuated by H₂ treatment. Hydrogen gas treatment inhibited LPS-induced pulmonary early and late NF-κB activation. Moreover, H₂ treatment dramatically prevented the LPS-induced pulmonary cell apoptosis in LPS-challenged mice, as reflected by the decrease in TUNEL (deoxynucleotidyl transferase dUTP nick end labeling) staining-positive cells and caspase 3 activity. Furthermore, H₂ treatment markedly attenuated LPS-induced lung neutrophil recruitment and inflammation, as evidenced by downregulation of lung myeloperoxidase activity, total cells, and polymorphonuclear neutrophils in BALF, as well as proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, interleukin 6, and high-mobility group box 1) and chemokines (keratinocyte-derived chemokine, macrophage inflammatory protein [MIP] 1α, MIP-2, and monocyte chemoattractant protein 1) in BALF. In addition, i.p. injection of 10 mL/kg hydrogen-rich saline also significantly attenuated the LPS-induced ALI. Collectively, these results demonstrate that molecular hydrogen treatment ameliorates LPS-induced ALI through reducing lung inflammation and apoptosis, which may be associated with the decreased NF-κB activity. Hydrogen gas may be useful as a novel therapy to treat ALI. munosorbent assay; H₂-hydrogen gas; HMGB1-high-mobility group box 1; HS-hydrogen-rich saline; i.t.-intratracheal; KC-keratinocyte-derived chemokine; LPS-lipopolysaccharide; MCP-1-monocyte chemoattractant protein 1; MIP-1α-macrophage inflammatory protein 1α; MIP-2-macrophage inflammatory protein 2; MPO-myeloperoxidase; PBS-phosphate-buffered saline; PMNs-polymorphonuclear neutrophils; TUNEL-deoxynucleotidyl transferase dUTP nick end labeling; W/D-wet-to-dry.


E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain.
The RNA-binding protein Hfq has been studied extensively for its function as a modulator of gene expression at the post-transcriptional level. While most Hfq studies have focused on the protein's interaction with sRNAs and mRNAs, Hfq binding to DNA has been observed but is less explored. During the isolation of Hfq from Escherichiacoli, we found genomic DNA fragments associated with the protein after multiple steps of purification. Sequences of 41 amplified segments from the DNA fragments associated with Hfq were determined. A large fraction of the DNA segments were predicted to have significant helical axis curvature and were from genes associated with membrane proteins, characteristics unexpected for non-specific binding. Analysis by analytical ultracentrifugation indicated that rA(18) binding to Hfq disrupts Hfq-DNA interactions. The latter observation suggests Hfq binding to DNA involves its distal surface. This was supported by a gel mobility shift assay that showed single amino acid mutations on the distal surface of Hfq inhibited Hfq binding to duplex DNA, while six of seven mutations on the proximal surface and outer circumference of the hexamer did not prevent Hfq binding. Two mutated Hfq which have portions of their C-terminal domain removed also failed to bind to DNA. The apparent K(d) for binding wild type Hfq to several duplex DNA was estimated from a gel mobility shift assay to be ~400nM.


Predicting Complete Remission of Acute Myeloid Leukemia: Machine Learning Applied to Gene Expression.
Machine learning (ML) is a useful tool for advancing our understanding of the patterns and significance of biomedical data. Given the growing trend on the application of ML techniques in precision medicine, here we present an ML technique which predicts the likelihood of complete remission (CR) in patients diagnosed with acute myeloid leukemia (AML). In this study, we explored the question of whether ML algorithms designed to analyze gene-expression patterns obtained through RNA sequencing (RNA-seq) can be used to accurately predict the likelihood of CR in pediatric AML patients who have received induction therapy. We employed tests of statistical significance to determine which genes were differentially expressed in the samples derived from patients who achieved CR after 2 courses of treatment and the samples taken from patients who did not benefit. We tuned classifier hyperparameters to optimize performance and used multiple methods to guide our feature selection as well as our assessment of algorithm performance. To identify the model which performed best within the context of this study, we plotted receiver operating characteristic (ROC) curves. Using the top 75 genes from the k-nearest neighbors algorithm (K-NN) model (K = 27) yielded the best area-under-the-curve (AUC) score that we obtained: 0.84. When we finally tested the previously unseen test data set, the top 50 genes yielded the best AUC = 0.81. Pathway enrichment analysis for these 50 genes showed that the guanosine diphosphate fucose (GDP-fucose) biosynthesis pathway is the most significant with an adjusted P value = .0092, which may suggest the vital role of N-glycosylation in AML.


Factors influencing endometrial thickness in postmenopausal women.
Cut-off values for endometrial thickness (ET) in asymptomatic postmenopausal woman have been standardized. However, there are no comprehensive studies to document how various factors can influence the ET after the age of menopause. To study the various factors influencing the ET in postmenopausal women. This was a prospective observational study. A total of 110 postmenopausal women underwent detailed history taking, clinical examination, and transvaginal scan for uterine volume and ovarian volume. The volumes were calculated by using ellipsoid formula: Width × thickness × height × 0.523. The variation in ET with respect to the influencing factors such as age, duration of menopause, parity, body mass index (BMI), medical illness like diabetes/hypertension, drugs like tamoxifen, presence of myoma, uterine volume, ovarian volume, and serum estradiol (in selected patients) were measured. Descriptive analysis was performed using SPSS software (version 16, Chicago II, USA) to obtain mean, standard deviation (SD), 95% confidence intervals (CIs) and inter quartile ranges. Comparison of means was carried out using analysis of variance. The mean (SD) age of the patients was 55.4 (6.91) years (95% CI, 54.1, 56.7). The mean (SD) age at menopause was 47.95 (3.90) years (95% CI, 47.2, 48.7) and the mean (SD) duration of menopause was 7.27 (6.65) years (95% CI, 6.01, 8.53). The mean (SD) ET was 3.8 (2.3) mm (95% CI, 3.36, 4.23). Medical illness like diabetes and hypertension did not alter the ET. ET increased as BMI increased and it was statistically significant. The presence of myoma increased uterine volume significantly and was associated with thick endometrial stripe. Similarly, whenever the ovaries were visualized and as the ovarian volume increased, there was an increase in ET. When ET was > 4 mm (n = 37), they were offered endocel, of which 16 agreed to undergo the procedure. None were found to have endometrial cancer. This study suggests that parity, BMI, presence of myoma, tamoxifen usage, uterine volume, ovarian volume and serum estradiol influence the ET in postmenopausal women.


Full-field dual-color 100-nm super-resolution imaging reveals organization and dynamics of mitochondrial and ER networks.
Most structured illumination microscopes use a physical or synthetic grating that is projected into the sample plane to generate a periodic illumination pattern. Albeit simple and cost-effective, this arrangement hampers fast or multi-color acquisition, which is a critical requirement for time-lapse imaging of cellular and sub-cellular dynamics. In this study, we designed and implemented an interferometric approach allowing large-field, fast, dual-color imaging at an isotropic 100-nm resolution based on a sub-diffraction fringe pattern generated by the interference of two colliding evanescent waves. Our all-mirror-based system generates illumination pat-terns of arbitrary orientation and period, limited only by the illumination aperture (NA = 1.45), the response time of a fast, piezo-driven tip-tilt mirror (10 ms) and the available fluorescence signal. At low µW laser powers suitable for long-period observation of life cells and with a camera exposure time of 20 ms, our system permits the acquisition of super-resolved 50 µm by 50 µm images at 3.3 Hz. The possibility it offers for rapidly adjusting the pattern between images is particularly advantageous for experiments that require multi-scale and multi-color information. We demonstrate the performance of our instrument by imaging mitochondrial dynamics in cultured cortical astrocytes. As an illustration of dual-color excitation dual-color detection, we also resolve interaction sites between near-membrane mitochondria and the endoplasmic reticulum. Our TIRF-SIM microscope provides a versatile, compact and cost-effective arrangement for super-resolution imaging, allowing the investigation of co-localization and dynamic interactions between organelles--important questions in both cell biology and neurophysiology.


Enumeration of beta-haemolytic streptococci on normal skin by direct agar contact.
Normal skin sites in children from a population in which streptococcal impetigo is endemic were examined for the presence of beta-haemolytic streptococci by a direct agar-contact technique. Ninety-eight of 554 samples (18%) were positive for these organisms. Penicillin prophylaxis reduced the frequency of isolation of streptococci from the normal skin for a period of 3 weeks, perhaps accounting in part for the lower isolation rate in this than in earlier studies. Numbers of streptococcal colony-forming units in positive samples were generally low, both in terms of absolute numbers isolated from the surface area sampled and in comparison with numbers of other aerobic flora recovered. The presence of streptococcal pyoderma at the time of agar contact was not necessarily associated with the presence of or with increased numbers of streptococci on samples obtained from normal skin sites. Low counts were consistently found in early summer and higher counts in some samples in late summer. In a simultaneous comparison of paired samples taken from adjacent sites, the frequency of detection of streptococci by direct agar contact compared favourably with that obtained with a moist-swab method. The increased frequency of detection by the agar-contact method appeared to be related to an increased sensitivity for the detection of low numbers of streptococcal colony-forming units on the normal skin.


Development of the cerebellum: simple steps to make a 'little brain'.
The cerebellum is a pre-eminent model for the study of neurogenesis and circuit assembly. Increasing interest in the cerebellum as a participant in higher cognitive processes and as a locus for a range of disorders and diseases make this simple yet elusive structure an important model in a number of fields. In recent years, our understanding of some of the more familiar aspects of cerebellar growth, such as its territorial allocation and the origin of its various cell types, has undergone major recalibration. Furthermore, owing to its stereotyped circuitry across a range of species, insights from a variety of species have contributed to an increasingly rich picture of how this system develops. Here, we review these recent advances and explore three distinct aspects of cerebellar development - allocation of the cerebellar anlage, the significance of transit amplification and the generation of neuronal diversity - each defined by distinct regulatory mechanisms and each with special significance for health and disease.


Continuous infusion of the anti-CD22 immunotoxin IgG-RFB4-SMPT-dgA in patients with B-cell lymphoma: a phase I study.
IgG-RFB4-SMPT-dgA consists of deglycosylated ricin A chain (dgA) coupled to the monoclonal antihuman CD22 antibody, RFB4. This study determined the maximally tolerated dose (MTD) of this immunotoxin (IT) administered as a continuous 8-day infusion to 18 patients with B-cell lymphoma (30% CD22+ tumor cells) over 8 days. The MTD was 19.2 mg/m2/192 h (maximum toxicity grade 1), with vascular leak syndrome (VLS) as dose-limiting toxicity (DLT) at 28.8 mg/m2/192 h (grades 3 through 5 in 7 of 11 patients). Predictors of severe VLS included serum IT concentrations greater than 1,000 ng/mL and the absence of circulating tumor cells. Decreased urine sodium excreted in 24 hours provided evidence for mild VLS without notable changes in serum albumin. Four partial responses, 3 minor responses, 6 stable disease, and 3 progression of disease were observed. The mean maximal serum concentration (Cmax) in initial courses at the MTD (19.2 mg/m2) was 443 +/- 144 ng/mL (n = 3; range, 326 to 604). At 28.8 mg/m2/192 h, the Cmax was highly variable (n = 11; mean, 1,102 +/- 702; range, 9.6 to 2,032 ng/mL). Human antimouse or antiricin antibodies developed in 6 of 16 (37.5%) patients after one course of IT. However, 10 eligible patients received multiple courses of IT. Changes in serum cytokines and cytokine receptors did not correlate with toxicity but decreased soluble interleukin-2 receptor concentrations correlated with clinical response. Comparison to a prior study with the same IT administered by intermittent bolus infusions (Amlot et al, Blood 82:2624, 1993) suggests similar clinical response, toxicity, and immunogenicity.