[Overweight and obesity: prevalence and determining social factors of overweight in the Peruvian population (2009-2010)].
Estimate the prevalence of overweight, obesity and the determining social factors of overweight in the Peruvian population. A cross-cut study was conducted which included family members from homes in the sample of the National Household Survey, 2009-2010. Stratified random and multistage sampling was used. The sample included 69 526 members; the anthropometric measurements were done based on the international methodology. To evaluate overweight and obesity, weight-for-height (children <5 years), BMI for age (children and teenagers from 5 to 19 years old), and BMI for adults were used. An analysis of complex samples was made in SPSS and the weighting factor was adjusted. Descriptive statistics and logistic regression were calculated with a 95% confidence interval. Overweight and obesity were higher in young adults (62.3%) and lower in children <5 years old (8.2%). The determining social factors for overweight according to age group were: not being poor (child <5 years old, children 5-9 years old, teenagers and the elderly), living in urban areas (child <5 years old, teenagers, young adults, adults and the elderly) and being a woman (children 5-9 years old, adults and the elderly). Overweight and obesity are indeed a public health issue in Peru. Not being poor and living in urban areas are determining social factors of overweight among Peruvian people.


[Evaluation of the clinical benefit of Permixon and tamsulosin in severe BPH patients--PERMAL study subset analysis].
To compare the efficacy of the lipido-sterolic extract of Serenoa repens, Permixon, to that of the a-blocker, tamsulosin, in the treatment of severe low urinary tract symptoms (LUTS) of benign prostatic hyperplasia (BPH). In a 12-month, double-blind, randomized study that showed equivalent efficacy of Permixon 320 mg/day and tamsulosin 0.4 mg/day ("PERMAL study"), 685 BPH patients with IPSS > 10 had been analyzed for efficacy. Of these, the 124 patients with severe LUTS (IPSS > 19) at randomization were retained for this subset analysis. After a 4-week run-in period, 59 and 65 patients had been randomized to tamsulosin and Permixon groups, respectively. Both treatment groups were compared regarding the evolution from baseline of total IPSS and its irritative and obstructive subscores. LUTS-related QpL, prostate volume, Qmax and MSF-4 (sexual activity questionnaire) at different time points over 1 year An analysis of variance of changes from baseline to end point was performed for all the parameters. The over-time evolutions of total, irritative and obstructive IPSS were further compared using a variance analysis for repeated measurements. At 12 months, total IPSS decreased by 7.8 with Permixon and 5.8 with tamsulosin (p = 0.051); the irritative symptoms improved significantly more (p = 0.049) with Permixon (- 2.9 versus - 1.9 with tamsulosin). The superiority of Permixon in reducing irritative symptoms appeared as soon as month 3 and was maintained up to month 12 (p = 0.03). Permixon 320 mg/day was shown to be slightly superior to tamsulosin 0.4 mg/day in reducing LUTS in severe BPH patients after 3 months and up to 12 months of treatment.


Role of rare variants in undetermined multiple adenomatous polyposis and early-onset colorectal cancer.
Some 15-20% of multiple adenomatous polyposis have no genetic explanation and 20-30% of colorectal cancer (CRC) cases are thought to be due to inherited multifactorial causes. Accumulation of deleterious effects of low-frequency dominant and independently acting variants may be a partial explanation for such patients. The aim of this study was to type a selection of rare and low-frequency variants (<5%) to elucidate their role in CRC susceptibility. A total of 1181 subjects were included (866 controls; 315 cases). Cases comprised UK (n=184) and French (n=131) patients with MAP (n=187) or early-onset CRC (n=128). Seventy variants in 17 genes were examined in cases and controls. The effect of the variant effect on protein function was investigated in silico. Out of the 70 variants typed, 36 (51%) were tested for association. Twenty-one variants were rare (minor allele frequency (MAF) <1%). Four rare variants were found to have a significantly higher MAF in cases (EXO1-12, MLH1-1, CTNNB1-1 and BRCA2-37, P<0.05) than in controls. Pooling all rare variants with a MAF <0.5% showed an excess risk in cases (odds ratio=3.2; 95% confidence interval=1.1-9.5; P=0.04). Rare variants are important risk factors in CRC and, as such, should be systematically assayed alongside common variation in the search for the genetic basis of complex diseases.


Systemic lupus erythematosus disease activity index 2000.
To describe the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K), a modification of SLEDAI to reflect persistent, active disease in those descriptors that had previously only considered new or recurrent occurrences, and to validate SLEDAI-2K against the original SLEDAI as a predictor for mortality and as a measure of global disease activity in the clinic. All visits in our cohort of 960 patients were used to correlate SLEDAI-2K against the original SLEDAI, and the whole cohort was used to validate SLEDAI-2K as a predictor of mortality. A subgroup of 212 patients with SLE followed at the Lupus Clinic who had 5 regular visits, 3-6 months apart, in 1991-93 was also included. An uninvolved clinician evaluated each patient record and assigned a clinical activity level. The SLEDAI score was calculated from the database according to both the original and modified definitions. SLEDAI-2K correlated highly (r = 0.97) with SLEDAI. Both methods for SLEDAI scoring predicted mortality equally (p = 0.0001), and described similarly the range of disease activity as recognized by the clinician. SLEDAI-2K, which allows for persistent activity in rash, mucous membranes, alopecia, and proteinuria, is suitable for use in clinical trials and studies of prognosis in SLE.


Effect of isoproterenol on myocardial perfusion, function, energy metabolism and nitric oxide pathway in the rat heart - a longitudinal MR study.
The chronic administration of the β-adrenoreceptor agonist isoproterenol (IsoP) is used in animals to study the mechanisms of cardiac hypertrophy and failure associated with a sustained increase in circulating catecholamines. Time-dependent changes in myocardial blood flow (MBF), morphological and functional parameters were assessed in rats in vivo using multimodal cardiac MRI. Energy metabolism, oxidative stress and the nitric oxide (NO) pathway were evaluated in isolated perfused rat hearts following 7 days of treatment. Male Wistar rats were infused for 7 days with IsoP or vehicle using osmotic pumps. Cine-MRI and arterial spin labeling were used to determine left ventricular morphology, function and MBF at days 1, 2 and 7 after pump implantation. Isolated hearts were then perfused, and high-energy phosphate compounds and intracellular pH were followed using ³¹P MRS with simultaneous measurement of contractile function. Total creatine and malondialdehyde (MDA) contents were measured by high-performance liquid chromatography. The NO pathway was evaluated by NO synthase isoform expression and total nitrate concentration (NO(x)). In IsoP-treated rats, left ventricular mass was increased at day 1 and maintained. Wall thickness was increased with a peak at day 2 and a tendency to return to baseline values at day 7. MBF was markedly increased at day 1 and returned to normal values between days 1 and 2. The rate-pressure product and phosphocreatine/adenosine triphosphate ratio in perfused hearts were reduced. MDA, endothelial NO synthase expression and NO(x) were increased. Sustained high cardiac function and normal MBF after 24 h of IsoP infusion indicate imbalance between functional demand and blood flow, leading to morphological changes. After 1 week, cardiac hypertrophy and decreased function were associated with impaired phosphocreatine, increased oxidative stress and up-regulation of the NO pathway. These results provide supplemental information on the evolution of the different contributing factors leading to morphological and functional changes in this model of cardiac hypertrophy and failure.


Randomized placebo-controlled study of the effects of simvastatin on haemostatic variables, lipoproteins and free fatty acids. The Oxford Cholesterol Study Group.
The Oxford Cholesterol Study is a randomized placebo-controlled trial designed primarily to assess the effects of simvastatin on blood cholesterol levels and side-effects in preparation for a large, long-term trial of the effects of cholesterol-lowering drug therapy on mortality. At present there is only limited evidence from randomized comparisons of the effects of HMG-CoA reductase inhibitors, such as simvastatin, on thrombogenic, as distinct from atherogenic, pathways in coronary heart disease. The present sub-study was carried out to assess the effects of simvastatin on a range of haemostatic variables, as well as on free fatty acids and on lipoprotein fractions not studied in detail previously. At an average of about 2 years after starting study treatment, non-fasting blood samples were obtained from a sequential sample of 162 participants who had been randomly allocated to receive 40 mg (54 patients) or 20 mg (57 patients) daily simvastatin or matching placebo treatment (51 patients). Only patients who reported taking their study treatment and who were not known to be diabetic or to be taking some other lipid lowering treatment were to be included. The principal comparisons were to be of those allocated simvastatin (i.e. 20 and 40 mg doses combined) vs those allocated placebo. Among patients allocated simvastatin, marginally significant lower factor VII antigen levels (12.10% +/- 6.08 of standard; 2P < 0.05) and non-significantly lower factor VII coagulant activity (8.24% +/- 4.99 of standard) and fibrinogen concentrations (0.10 +/- 0.08 g. l-1) were observed. In contrast, plasminogen activator inhibitor activity was significantly higher (2.62 +/- 1.03 IU; 2P < 0.01) among patients allocated simvastatin. No significant differences were seen in the other haemostatic factors studied (e.g. prothrombin fragment 1.2, factor XII and C1 inhibitor). Total free fatty acid concentration was marginally significantly reduced (2P = 0.02) with simvastatin, but none of the reductions in individual free fatty acids was significant. Lipoprotein fractions were only measured among patients allocated 40 mg daily simvastatin or placebo. Compared with placebo, simvastatin produced significant decreases not only in LDL cholesterol (1.74 +/- 0.15 mmol.1(-1): 2P < 0.0001) but also in VLDL cholesterol (0.28 +/- 0.08 mmol.1(-1); 2P < 0.001) and IDL cholesterol (0.17 +/- 0.03 mmol.1(-1); 2P < 0.0001). There were also lower triglyceride levels associated with LDL (0.07 +/- 0.01 mmol.1(-1); 2P < 0.0001), IDL (0.03 +/- 0.01 mmol.1(-1); 2P < 0.01) and VLDL (0.27 +/- 0.14; 2P = 0.05). The effects of simvastatin on haemostatic variables appear to be far less marked than its lipid effects. Given the associations of haemostatic factors with coronary heart disease incidence, larger randomized comparisons of the HMG-CoA reductase inhibitors (and of the newer fibrates which may produce greater effects) are needed to provide more reliable estimates of the extent to which they influence these variables.


Calibration of a high spectral resolution lidar using a Michelson interferometer, with data examples from ORACLES.
The NASA Langley airborne second-generation High Spectral Resolution Lidar (HSRL-2) uses a density-tuned field-widened Michelson interferometer to implement the HSRL technique at 355 nm. The Michelson interferometer optically separates the received backscattered light between two channels, one of which is dominated by molecular backscattering, while the other contains most of the light backscattered by particles. This interferometer achieves high and stable contrast ratio, defined as the ratio of particulate backscatter signal received by the two channels. We show that a high and stable contrast ratio is critical for precise and accurate backscatter and extinction retrievals. Here, we present retrieval equations that take into account the incomplete separation of particulate and molecular backscatter in the measurement channels. We also show how the accuracy of the contrast ratio assessment propagates to error in the optical properties. For both backscattering and extinction, larger errors are produced by underestimates of the contrast ratio (compared to overestimates), more extreme aerosol loading, and-most critically-smaller true contrast ratios. We show example results from HSRL-2 aboard the NASA ER-2 aircraft from the 2016 ORACLES field campaign in the southeast Atlantic, off the coast of Africa, during the biomass burning season. We include a case study where smoke aerosol in two adjacent altitude layers showed opposite differences in extinction- and backscatter-related Ångström exponents and a reversal of the lidar ratio spectral dependence, signatures which are shown to be consistent with a relatively modest difference in smoke particle size.


Diet in dermatology: revisited.
Diet has an important role to play in many skin disorders, and dermatologists are frequently faced with the difficulty of separating myth from fact when it comes to dietary advice for their patients. Patients in India are often anxious about what foods to consume, and what to avoid, in the hope that, no matter how impractical or difficult this may be, following this dictum will cure their disease. There are certain disorders where one or more components in food are central to the pathogenesis, e.g. dermatitis herpetiformis, wherein dietary restrictions constitute the cornerstone of treatment. A brief list, although not comprehensive, of other disorders where diet may have a role to play includes atopic dermatitis, acne vulgaris, psoriasis vulgaris, pemphigus, urticaria, pruritus, allergic contact dermatitis, fish odor syndrome, toxic oil syndrome, fixed drug eruption, genetic and metabolic disorders (phenylketonuria, tyrosinemia, homocystinuria, galactosemia, Refsum's disease, G6PD deficiency, xanthomas, gout and porphyria), nutritional deficiency disorders (kwashiorkar, marasmus, phrynoderma, pellagra, scurvy, acrodermatitis enteropathica, carotenemia and lycopenemia) and miscellaneous disorders such as vitiligo, aphthous ulcers, cutaneous vasculitis and telogen effluvium. From a practical point of view, it will be useful for the dermatologist to keep some dietary information handy to deal with the occasional patient who does not seem to respond in spite of the best, scientific and evidence-based therapy.


Is it time to shift to better characterization of patients in trials assessing novel antidepressants? An example of two relapse prevention studies with agomelatine.
The present paper reports in parallel the findings of the two studies that evaluated the efficacy of agomelatine in preventing relapse of depression. It describes the methodological adjustments made between the first and the second trial, particularly in relation to patient selection and accuracy of diagnosis of depression. Patients with major depressive disorder who responded to an 8/10-week course of agomelatine 25-50 mg treatment were randomly assigned to receive continuation treatment with agomelatine or placebo during a 24-week, randomized, double-blind treatment period with an optional 18- or 20-week double-blind extension period. The cumulative probability of relapse was calculated using the Kaplan-Meier method of survival analysis. Study 1 lacked assay sensitivity because of an unexpectedly low relapse rate in the placebo arm, but was instructive in showing that the agomelatine effect was better than placebo only in those patients with higher symptom levels at baseline. Study 2 showed a robust benefit of agomelatine - a two-fold reduction in the relapse rate - observed at least up to 10 months in both the overall population and the more severely depressed patients. The methodological adjustments introduced in study 2 (e.g. a minimum subscore calculated from eight specific Hamilton Depression Rating Scale items, the use of the self-rating questionnaire Hospital Anxiety Depression Scale and the Sheehan questionnaire) have assured an adequate severity of depression not only on the basis of ratings of symptom severity but also on measures of functional impairment. We did not find increased severity of symptoms in study 2, but we hypothesize that the increased demands on investigators improved the quality of recruitment to represent more real-world patients. Adopting these innovations could contribute towards lower failure rates for future placebo-controlled clinical trials in the field.


Imaging opioid analgesia in the human brain and its potential relevance for understanding opioid use in chronic pain.
Opioids play an important role for the management of acute pain and in palliative care. The role of long-term opioid therapy in chronic non-malignant pain remains unclear and is the focus of much clinical research. There are concerns regarding analgesic tolerance, paradoxical pain and issues with dependence that can occur with chronic opioid use in the susceptible patient. In this review, we discuss how far human neuroimaging research has come in providing a mechanistic understanding of pain relief provided by opioids, and suggest avenues for further studies that are relevant to the management of chronic pain with opioids. This article is part of the Special Issue Section entitled 'Neuroimaging in Neuropharmacology'.


The cerebral response during subjective choice with and without self-reference.
The anterior medial prefrontal (AMPFC) and retrosplenial (RSC) cortices are active during self-referential decision-making tasks such as when participants appraise traits and abilities, or current affect. Other appraisal tasks requiring an evaluative decision or mental representation, such as theory of mind and perspective-taking tasks, also involve these regions. In many instances, these types of decisions involve a subjective opinion or preference, but also a degree of ambiguity in the decision, rather than a strictly veridical response. However, this ambiguity is generally not controlled for in studies that examine self-referential decision-making. In this functional magnetic resonance imaging experiment with 17 healthy adults, we examined neural processes associated with subjective decision-making with and without an overt self-referential component. The task required subjective decisions about colors-regarding self-preference (internal subjective decision) or color similarity (external subjective decision) under conditions where there was no objectively correct response. Results indicated greater activation in the AMPFC, RSC, and caudate nucleus during internal subjective decision-making. The findings suggest that self-referential processing, rather than subjective judgments among ambiguous response alternatives, accounted for the AMPFC and RSC response.


Modulation of human extrastriate visual processing by selective attention to colours and words.
The present study investigated the effect of visual selective attention upon neural processing within functionally specialized regions of the human extrastriate visual cortex. Field potentials were recorded directly from the inferior surface of the temporal lobes in subjects with epilepsy. The experimental task required subjects to focus attention on words from one of two competing texts. Words were presented individually and foveally. Texts were interleaved randomly and were distinguishable on the basis of word colour. Focal field potentials were evoked by words in the posterior part of the fusiform gyrus. Selective attention strongly modulated long-latency potentials evoked by words. The attention effect co-localized with word-related potentials in the posterior fusiform gyrus, and was independent of stimulus colour. The results demonstrated that stimuli receive differential processing within specialized regions of the extrastriate cortex as a function of attention. The late onset of the attention effect and its co-localization with letter string-related potentials but not with colour-related potentials recorded from nearby regions of the fusiform gyrus suggest that the attention effect is due to top-down influences from downstream regions involved in word processing.


Autophagy-Dependent Generation of Free Fatty Acids Is Critical for Normal Neutrophil Differentiation.
Neutrophils are critical and short-lived mediators of innate immunity that require constant replenishment. Their differentiation in the bone marrow requires extensive cytoplasmic and nuclear remodeling, but the processes governing these energy-consuming changes are unknown. While previous studies show that autophagy is required for differentiation of other blood cell lineages, its function during granulopoiesis has remained elusive. Here, we have shown that metabolism and autophagy are developmentally programmed and essential for neutrophil differentiation in vivo. Atg7-deficient neutrophil precursors had increased glycolytic activity but impaired mitochondrial respiration, decreased ATP production, and accumulated lipid droplets. Inhibiting autophagy-mediated lipid degradation or fatty acid oxidation alone was sufficient to cause defective differentiation, while administration of fatty acids or pyruvate for mitochondrial respiration rescued differentiation in autophagy-deficient neutrophil precursors. Together, we show that autophagy-mediated lipolysis provides free fatty acids to support a mitochondrial respiration pathway essential to neutrophil differentiation.


Sertraline versus other antidepressive agents for depression.
The National Institute for Health and Clinical Excellence clinical practice guideline on the treatment of depressive disorder recommended that selective serotonin reuptake inhibitors should be the first-line option when drug therapy is indicated for a depressive episode. Preliminary evidence suggested that sertraline might be slightly superior in terms of effectiveness. To assess the evidence for the efficacy, acceptability and tolerability of sertraline in comparison with tricyclics (TCAs), heterocyclics, other SSRIs and newer agents in the acute-phase treatment of major depression. MEDLINE (1966 to 2008), EMBASE (1974 to 2008), the Cochrane Collaboration Depression, Anxiety and Neurosis Controlled Trials Register and the Cochrane Central Register of Controlled Trials up to July 2008. No language restriction was applied. Reference lists of relevant papers and previous systematic reviews were hand-searched. Pharmaceutical companies and experts in this field were contacted for supplemental data. Randomised controlled trials allocating patients with major depression to sertraline versus any other antidepressive agent. Two review authors independently extracted data. Discrepancies were resolved with another member of the team. A double-entry procedure was employed by two reviewers. Information extracted included study characteristics, participant characteristics, intervention details and outcome measures in terms of efficacy (the number of patients who responded or remitted), acceptability (the number of patients who failed to complete the study) and tolerability (side-effects). A total of 59 studies, mostly of low quality, were included in the review, involving multiple treatment comparisons between sertraline and other antidepressant agents. Evidence favouring sertraline over some other antidepressants for the acute phase treatment of major depression was found, either in terms of efficacy (fluoxetine) or acceptability/tolerability (amitriptyline, imipramine, paroxetine and mirtazapine). However, some differences favouring newer antidepressants in terms of efficacy (mirtazapine) and acceptability (bupropion) were also found. In terms of individual side effects, sertraline was generally associated with a higher rate of participants experiencing diarrhoea. This systematic review and meta-analysis highlighted a trend in favour of sertraline over other antidepressive agents both in terms of efficacy and acceptability, using 95% confidence intervals and a conservative approach, with a random effects analysis. However, the included studies did not report on all the outcomes that were pre-specified in the protocol of this review. Outcomes of clear relevance to patients and clinicians were not reported in any of the included studies.


Outdoor air pollutants and patient health.
Almost 160 million persons live in areas of the United States that exceed federal health-based air pollution standards. The two air pollutants that most commonly exceed standards are ozone and particulate matter. Ozone and particulate matter can harm anyone if levels are sufficiently elevated, but health risk from air pollution is greatest among vulnerable populations. Both ozone and particulate matter can cause pulmonary inflammation, decreased lung function, and exacerbation of asthma and chronic obstructive pulmonary disease. Particulate matter is also strongly associated with increased cardiovascular morbidity and mortality. Children, older adults, and other vulnerable persons may be sensitive to lower levels of air pollution. Persons who are aware of local air pollution levels, reported daily by the U.S. Environmental Protection Agency as the Air Quality Index, can take action to reduce exposure. These actions include simple measures to limit exertion and time spent outdoors when air pollution levels are highest, and to reduce the infiltration of outdoor air pollutants into indoor spaces.


Prevalence of overweight and obesity in young Greek men.
We determined the prevalence of overweight and obesity in young Greek men in 2006 and examined variations related to their place of residence and educational level. Body height and weight were measured in 2568 conscripts of the Greek army, aged 19-26 years. The calculated body mass index (BMI, kg m(-2)) was correlated to their socio-demographic characteristics, i.e. level of education and place of residence (urban or rural). Overweight and obesity were defined according to the World Health Organization classification. Mean BMI (standard deviation) of the conscripts was 24.7 (4.2). The prevalence of overweight (30 > BMI > or = 25 kg m(-2)) was 28.5% and correlated positively with a higher educational level, whereas the prevalence of obesity (BMI > or = 30 kg m(-2)) was 10.4% and correlated positively with a lower educational level. Our data were compared with those of similar studies performed in the years 1969: BMI 23.8 (1.4) (P < 0.0001) and 1990: BMI 23.8 (2.9) (P < 0.0001), showing a positive secular trend for BMI in Greek conscripts in the last 16 years. In conclusion, we documented an alarmingly high prevalence of overweight and obesity among young Greek men.


Autoantibodies in senear-usher syndrome: cross-reactivity or multiple autoimmunity?
Senear-Usher syndrome or pemphigus erythematosus is a pathology that overlaps clinically and serologically with pemphigus foliaceus and lupus erythematosus. Skin biopsies of patients with pemphigus erythematosus reveal acantholysis and deposits of immunoglobulins in desmosomes, and they are positive in the lupus band test. In the present paper, we determined whether the autoantibodies associated with pemphigus erythematosus targeted a single antigen or multiple antigens as a result of the stimulation of independent B cell clones. Our present paper demonstrates that patients with pemphigus erythematosus produce both antiepithelial antibodies specific for desmoglein 1 and 3 and antinuclear antibodies specific for Ro, La, Sm, and double-stranded DNA antigens. After eluting specific anti-epithelial or anti-nuclear antibodies, which were recovered and tested using double-fluorescence assays, a lack of cross-reactivity was demonstrated between desmosomes and nuclear and cytoplasmic lupus antigens. This result suggests that autoantibodies in pemphigus erythematosus are directed against different antigens and that these autoantibodies are produced by independent clones. Given these clinical and serological data, we suggest that pemphigus erythematosus behaves as a multiple autoimmune disease.


A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.
Many human melanoma tumors express antigens that are recognized in vitro by cytolytic T lymphocytes (CTLs) derived from the tumor-bearing patient. A gene was identified that directed the expression of antigen MZ2-E on a human melanoma cell line. This gene shows no similarity to known sequences and belongs to a family of at least three genes. It is expressed by the original melanoma cells, other melanoma cell lines, and by some tumor cells of other histological types. No expression was observed in a panel of normal tissues. Antigen MZ2-E appears to be presented by HLA-A1; anti-MZ2-E CTLs of the original patient recognized two melanoma cell lines of other HLA-A1 patients that expressed the gene. Thus, precisely targeted immunotherapy directed against antigen MZ2-E could be provided to individuals identified by HLA typing and analysis of the RNA of a small tumor sample.


Effects of dielectric relaxation on the director dynamics of uniaxial nematic liquid crystals.
The dielectric anisotropy of liquid crystals causes director reorientation in an applied electric field and is thus at the heart of electro-optic applications of these materials. The components of the dielectric tensor are frequency dependent. Until recently, this frequency dependence was not accounted for in a description of director dynamics in an electric field. We theoretically derive the reorienting dielectric torque acting on the director, taking into account the entire frequency spectrum of the dielectric tensor. The model allows one to include the effects of multiple relaxations in both parallel and perpendicular components of the dielectric tensor, thus generalizing a recent model [Y. Yin, Phys. Rev. Lett. 95, 087801 (2005)] limited by the single-relaxation approach. The model predicts the "dielectric memory effect" (DME)--i.e., dependence of the dielectric torque on both the "present" and "past" values of the electric field and the director. The model describes the experimentally observed director reorientation in the case when the rise time of the applied voltage is smaller than the dielectric relaxation time. In typical materials such as pentylcyanobiphenyl (5CB), in which the dielectric anisotropy is positive at low frequencies, the DME slows down the director reorientation in a sharply rising electric field, as the sharp front is perceived as a high-frequency excitation for which the dielectric anisotropy is small or even of a negative sign. In materials that are dielectrically negative, the DME speeds up the response when a sharp pulse is applied.


Immune cell census in murine atherosclerosis: cytometry by time of flight illuminates vascular myeloid cell diversity.
Atherosclerosis is characterized by the abundant infiltration of myeloid cells starting at early stages of disease. Myeloid cells are key players in vascular immunity during atherogenesis. However, the subsets of vascular myeloid cells have eluded resolution due to shared marker expression and atypical heterogeneity in vascular tissues. We applied the high-dimensionality of mass cytometry to the study of myeloid cell subsets in atherosclerosis. Apolipoprotein E-deficient (ApoE-/-) mice were fed a chow or a high fat (western) diet for 12 weeks. Single-cell aortic preparations were probed with a panel of 35 metal-conjugated antibodies using cytometry by time of flight (CyTOF). Clustering of marker expression on live CD45+ cells from the aortas of ApoE-/- mice identified 13 broad populations of leucocytes. Monocyte, macrophage, type 1 and type 2 conventional dendritic cell (cDC1 and cDC2), plasmacytoid dendritic cell (pDC), neutrophil, eosinophil, B cell, CD4+ and CD8+ T cell, γδ T cell, natural killer (NK) cell, and innate lymphoid cell (ILC) populations accounted for approximately 95% of the live CD45+ aortic cells. Automated clustering algorithms applied to the Lin-CD11blo-hi cells revealed 20 clusters of myeloid cells. Comparison between chow and high fat fed animals revealed increases in monocytes (both Ly6C+ and Ly6C-), pDC, and a CD11c+ macrophage subset with high fat feeding. Concomitantly, the proportions of CD206+ CD169+ subsets of macrophages were significantly reduced as were cDC2. A CyTOF-based comprehensive mapping of the immune cell subsets within atherosclerotic aortas from ApoE-/- mice offers tools for myeloid cell discrimination within the vascular compartment and it reveals that high fat feeding skews the myeloid cell repertoire toward inflammatory monocyte-macrophage populations rather than resident macrophage phenotypes and cDC2 during atherogenesis.


Chromatin immunoprecipitation from human embryonic stem cells.
The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones (4). Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors. Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation. Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.


Amygdalar interhemispheric functional connectivity differs between the non-depressed and depressed human brain.
The amygdalae are important, if not critical, brain regions for many affective, attentional and memorial processes, and dysfunction of the amygdalae has been a consistent finding in the study of clinical depression. Theoretical models of the functional neuroanatomy of both normal and psychopathological affective processes which posit cortical hemispheric specialization of functions have been supported by both lesion and functional neuroimaging studies in humans. Results from human neuroimaging studies in support of amygdalar hemispheric specialization are inconsistent. However, recent results from human lesion studies are consistent with hemispheric specialization. An important, yet largely ignored, feature of the amygdalae in the primate brain--derived from both neuroanatomical and electrophysiological data--is that there are virtually no direct interhemispheric connections via the anterior commissure (AC). This feature stands in stark contrast to that of the rodent brain wherein virtually all amygdalar nuclei have direct interhemispheric connections. We propose this feature of the primate brain, in particular the human brain, is a result of influences from frontocortical hemispheric specialization which have developed over the course of primate brain evolution. Results consistent with this notion were obtained by examining the nature of human amygdalar interhemispheric connectivity using both functional magnetic resonance imaging (FMRI) and positron emission tomography (PET). We found modest evidence of amygdalar interhemispheric functional connectivity in the non-depressed brain, whereas there was strong evidence of functional connectivity in the depressed brain. We interpret and discuss the nature of this connectivity in the depressed brain in the context of dysfunctional frontocortical-amygdalar interactions which accompany clinical depression.


Functions, structure, and read-through alternative splicing of feline APOBEC3 genes.
Over the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene. Here we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by read-through alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection. Our data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher.


Sparse whole-genome sequencing identifies two loci for major depressive disorder.
Major depressive disorder (MDD), one of the most frequently encountered forms of mental illness and a leading cause of disability worldwide, poses a major challenge to genetic analysis. To date, no robustly replicated genetic loci have been identified, despite analysis of more than 9,000 cases. Here, using low-coverage whole-genome sequencing of 5,303 Chinese women with recurrent MDD selected to reduce phenotypic heterogeneity, and 5,337 controls screened to exclude MDD, we identified, and subsequently replicated in an independent sample, two loci contributing to risk of MDD on chromosome 10: one near the SIRT1 gene (P = 2.53 × 10(-10)), the other in an intron of the LHPP gene (P = 6.45 × 10(-12)). Analysis of 4,509 cases with a severe subtype of MDD, melancholia, yielded an increased genetic signal at the SIRT1 locus. We attribute our success to the recruitment of relatively homogeneous cases with severe illness.


One-eyed stereo: a general approach to modeling 3-d scene geometry.
A single two-dimensional image is an ambiguous representation of the three-dimensional world¿many different scenes could have produced the same image¿yet the human visual system is ex-tremely successful at recovering a qualitatively correct depth model from this type of representation. Workers in the field of computational vision have devised a number of distinct schemes that attempt to emulate this human capability; these schemes are collectively known as ``shape from...'' methods (e.g., shape from shading, shape from texture, or shape from contour). In this paper we contend that the distinct assumptions made in each of these schemes is tantamount to providing a second (virtual) image of the original scene, and that each of these approaches can be translated into a conventional stereo formalism. In particular, we show that it is frequently possible to structure the problem as one of recovering depth from a stereo pair consisting of the supplied perspective image (the original image) and an hypothesized orthographic image (the virtual image). We present a new algorithm of the form required to accomplish this type of stereo reconstruction task.


Efficacy and safety of Fufangkushen colon-coated capsule in the treatment of ulcerative colitis compared with mesalazine: a double-blinded and randomized study.
Fufangkushen colon-coated capsule (FCC) was a newly developed herbal drug for treating ulcerative colitis (UC) patients with traditional Chinese medicine (TCM) pattern of damp-heat accumulating in the interior. To explore the efficacy and safety of FCC in the treatment of active UC compared with Huidi (HD, mesalazine enteric-coated tablets) were evaluated in a double-blinded and randomized clinical trial. In the double-blind, double-dummy, multicenter, randomized and controlled study, 320 active UC patients with TCM pattern of damp-heat accumulating in the interior were assigned to two groups: 240 treated with FCC plus HD placebo treatment, 80 with HD plus FCC placebo. The drugs and their corresponding placebos were administrated at advised dosage for 8 weeks. The primary endpoint was a positive clinical response at week 8, and Mayo scoring system was employed for assessment of UC activity. At the 8th week, 72.50% of patients in FCC group (170 of 234) and 65.00% of patients in HD group (52 of 80) had achieved a clinical response. There was no statistically significance between the 2 groups (P>0.05). The proportions of patients who had a clinical remission was similar in 2 groups (41.50% in FCC group, 41.25% in HD group, P>0.05), mucosal healing rate at week 8 in the two groups were also without significant difference (55.13% in FCC group, 55.00% in HD group, P>0.05). Mayo scores at week 8 showed no statistically difference in the two groups. No significant differences were observed between the safety profiles of the 2 groups (P>0.05). No severe AEs were reported in either group. The latent class analysis indicated that FCC was superior applicable for the left hemicolon involved patients than HD. Compared with HD, a mesalamine enteric-coated tablet, FCC is similarly effective and safe in the treatment of active UC with TCM pattern of damp-heat accumulation interior pattern. In addition, FCC indicates superior effect in the treatment of UC with inflamed area of the left hemicolon than HD.


Increased 4R tau expression and behavioural changes in a novel MAPT-N296H genomic mouse model of tauopathy.
The microtubule-associated protein tau is implicated in various neurodegenerative diseases including Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration, which are characterized by intracellular accumulation of hyperphosphorylated tau. Mutations in the tau gene MAPT cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). In the human central nervous system, six tau isoforms are expressed, and imbalances in tau isoform ratios are associated with pathology. To date, few animal models of tauopathy allow for the potential influence of these protein isoforms, relying instead on cDNA-based transgene expression. Using the P1-derived artificial chromosome (PAC) technology, we created mouse lines expressing all six tau isoforms from the human MAPT locus, harbouring either the wild-type sequence or the disease-associated N296H mutation on an endogenous Mapt-/- background. Animals expressing N296H mutant tau recapitulated early key features of tauopathic disease, including a tau isoform imbalance and tau hyperphosphorylation in the absence of somatodendritic tau inclusions. Furthermore, N296H animals displayed behavioural anomalies such as hyperactivity, increased time in the open arms of the elevated plus maze and increased immobility during the tail suspension test. The mouse models described provide an excellent model to study the function of wild-type or mutant tau in a highly physiological setting.


KIR3DL2 binds to HLA-B27 dimers and free H chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis.
The human leukocyte Ag HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of APC as both classical β2-microglobulin-associated B27 and B27 free H chain forms (FHC), including disulfide-bonded H chain homodimers (termed B27(2)). B27 FHC forms, but not classical B27, bind to KIR3DL2. HLA-A3, which is not associated with spondyloarthritis (SpA), is also a ligand for KIR3DL2. In this study, we show that B27(2) and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B27(2) tetramers bound KIR3DL2-transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric, and monomeric FHC from HLA-B27-expressing cell lines. Binding to B27(2) and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B27(2) and B27 FHC stimulated KIR3DL2CD3ε-transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFN-γ secretion and promoted greater survival of KIR3DL2(+) CD4 T and NK cells than binding to other HLA-class I. KIR3DL2(+) T cells from B27(+) SpA patients proliferated more in response to Ag presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27(+) SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.


Novel brain imaging approaches to understand acquired and congenital neuro-ophthalmological conditions.
The arrival of large datasets and the on-going refinement of neuroimaging technology have led to a number of recent advances in our understanding of visual pathway disorders. This work can broadly be classified into two areas, both of which are important when considering the optimal management strategies. The first looks at the delineation of damage, teasing out subtle changes to (specific components of) the visual pathway, which may help evaluate the severity and extent of disease. The second uses neuroimaging to investigate neuroplasticity, via changes in connectivity, cortical thickness, and retinotopic maps within the visual cortex. Here, we give consideration to both acquired and congenital patients with damage to the visual pathway, and how they differ. Congenital disorders of the peripheral visual system can provide insight into the large-scale reorganization of the visual cortex: these are investigated with reference to disorders of the optic chiasm and anophthalmia (absence of the eyes). In acquired conditions, we consider the recent work describing patterns of degeneration, both following single insult and in neurodegenerative conditions. We also discuss the developments in functional neuroimaging, with particular reference to work on hemianopia and the controversial suggestion of cortical reorganization following acquired retinal injury. Techniques for comparing neuro-ophthalmological conditions with healthy visual systems provide sensitive metrics to uncover subtle differences in grey and white matter structure of the brain. It is now possible to compare the massive reorganization present in congenital conditions with the apparent lack of plasticity following acquired damage.


Integrating human indoor air pollutant exposure within Life Cycle Impact Assessment.
Neglecting health effects from indoor pollutant emissions and exposure, as currently done in Life Cycle Assessment (LCA), may result in product or process optimizations at the expense of workers' or consumers' health. To close this gap, methods for considering indoor exposure to chemicals are needed to complement the methods for outdoor human exposure assessment already in use. This paper summarizes the work of an international expert group on the integration of human indoor and outdoor exposure in LCA, within the UNEP/ SETAC Life Cycle Initiative. A new methodological framework is proposed for a general procedure to include human-health effects from indoor exposure in LCA. Exposure models from occupational hygiene and household indoor air quality studies and practices are critically reviewed and recommendations are provided on the appropriateness of various model alternatives in the context of LCA. A single-compartment box model is recommended for use as a default in LCA, enabling one to screen occupational and household exposures consistent with the existing models to assess outdoor emission in a multimedia environment. An initial set of model parameter values was collected. The comparison between indoor and outdoor human exposure per unit of emission shows that for many pollutants, intake per unit of indoor emission may be several orders of magnitude higher than for outdoor emissions. It is concluded that indoor exposure should be routinely addressed within LCA.


Feasibility and parent satisfaction of a physical therapy intervention program for children with acute lymphoblastic leukemia in the first 6 months of medical treatment.
Children with acute lymphoblastic leukemia (ALL) are at risk for developing musculoskeletal complications during and after their medical treatment. The objective of this study was to examine the feasibility of an in-hospital physical therapy- and home exercise program during the first four phases of medical treatment, for children with newly diagnosed ALL. Nine patients, between the ages of 2-14 years old were enrolled within 2 weeks of diagnosis in the study. Each patient was evaluated at study entry, after each of the first four phases of therapy and each time patients were re-admitted to the hospital. Following the initial physical therapy evaluation an individualized home exercise program was developed, consisting of stretching, strengthening, and aerobic exercises. The following end points were measured at each evaluation: gross motor assessment as measured by gross motor function measure (GMFM), health-related quality of life as measured by the PedsQL and parent satisfaction questionnaire. This study was feasible with 98% of the evaluation sessions completed. The GMFM and PedsQL improved steadily throughout the study; however, the PedsQL slightly decreased from interim maintenance to delayed intensification. The parents reported being satisfied with the physical therapy program. We demonstrated that an in-hospital- and home exercise physical therapy program during the first four phases of medical treatments is feasible for children with ALL. Future randomized studies are needed to confirm whether an initial physical therapy program at diagnosis in children with ALL will limit functional impairments, improve overall fitness and increase health-related quality of life.


The contribution of the acute phase response to the pathogenesis of relapse in chronic-relapsing experimental autoimmune encephalitis models of multiple sclerosis.
Increased relapse rates in multiple sclerosis (MS) as a consequence of peripheral immune system activation, owing to infection for example, have been widely reported, but the mechanism remains unclear. Acute brain injury models can be exacerbated by augmenting the hepatic acute phase response (APR). Here, we explored the contribution of the hepatic APR to relapse in two rodent models of MS. Mice with MOG-CFA-induced chronic relapsing experimental autoimmune encephalitis (CR-EAE) were killed before, during and after the first phase of disease, and the brain and liver chemokine, cytokine and acute phase protein (APP) mRNA expression profile was determined. During remission, the APR was reactivated with an intraperitoneal lipopolysaccharide (LPS) and clinical score was monitored throughout. To explore the downstream mediators, CXCL-1, which is induced as part of the APR, was injected into animals with a focal, cytokine/MOG-induced EAE lesion (fEAE) and the cellularity of the lesions was assessed. Compared to CFA control, in a rodent CR-EAE model, an hepatic APR preceded clinical signs and central cytokine production in the initial phase of disease. Compared to administration in naïve animals, an LPS challenge during the asymptomatic remission phase of CR-EAE rodents provoked relapse and resulted in the increased and extended expression of specific peripheral hepatic chemokines. CXCL-1 and several other APPs were markedly elevated. A single intravenous administration of the highly induced chemokine, CXCL-1, was found to be sufficient to reactivate the lesions by increasing microglial activation and the recruitment of T cells in fEAE lesions. The APR plays a contributing role to the pathology seen in models of chronic brain injury and in translating the effects of peripheral immune system stimulation secondary to trauma or infection into central pathology and behavioural signs. Further elucidation of the exact mechanisms in this process will inform development of more effective, selective therapies in MS that, by suppressing the hepatic chemokine response, may prevent relapse.


beta-blocker binding to human 5-HT(1A) receptors in vivo and in vitro: implications for antidepressant therapy.
A novel strategy for improving the treatment of depressive illness is augmentation of antidepressants with a 5-HT1(1A) autoreceptor antagonist. However, trials using the 5-HT1(1A)/beta-blocker pindolol are proving inconsistent. We report how positron emission tomography (PET) and in vitro autoradiography can inform trials of antidepressant augmentation. We show that in healthy volunteers, in vivo, pindolol (n = 10) and penbutolol (n = 4), but not tertatolol (n = 4) occupy the human 5-HT(1A) receptors, at clinical doses. Pindolol, as well as the beta-blockers penbutolol and tertatolol, has high affinity for human 5-HT(1A) receptors in post-mortem brain slices (n = 4). Pindolol shows preference for 5-HT(1A) autoreceptors versus the post-synaptic receptors both in vitro and in vivo. Our data reveal that pindolol doses used in antidepressant trials so far are suboptimal for significant occupancy at the 5-HT(1A) autoreceptor. Penbutolol or higher doses of pindolol are candidates for testing as antidepressant augmenting regimes in future clinical trials.


Enhancing clinical evidence by proactively building quality into clinical trials.
Stakeholders across the clinical trial enterprise have expressed concern that the current clinical trial enterprise is unsustainable. The cost and complexity of trials have continued to increase, threatening our ability to generate reliable evidence essential for making appropriate decisions concerning the benefits and harms associated with clinical interventions. Overcoming this inefficiency rests on improving protocol design, trial planning, and quality oversight. The Clinical Trials Transformation Initiative convened a project to evaluate methods to prospectively build quality into the scientific and operational design of clinical trials ("quality-by-design"), such that trials are feasible to conduct and important errors are prevented rather than remediated. A working group evaluated aspects of trial design and oversight and developed the Clinical Trials Transformation Initiative quality-by-design principles document, outlining a series of factors generally relevant to the reliability of trial conclusions and to patient safety. These principles were then applied and further refined during a series of hands-on workshops to evaluate their utility in facilitating proactive, cross-functional dialogue, and decision-making about trial design and planning. Following these workshops, independent qualitative interviews were conducted with 19 workshop attendees to explore the potential challenges for implementing a quality-by-design approach to clinical trials. The Clinical Trials Transformation Initiative project team subsequently developed recommendations and an online resource guide to support implementation of this approach. The Clinical Trials Transformation Initiative quality-by-design principles provide a framework for assuring that clinical trials adequately safeguard participants and provide reliable information on which to make decisions on the effects of treatments. The quality-by-design workshops highlighted the value of active discussions incorporating the different perspectives within and external to an organization (e.g. clinical investigators, research site staff, and trial participants) in improving trial design. Workshop participants also recognized the value of focusing oversight on those aspects of the trial where errors would have a major impact on participant safety and reliability of results. Applying the Clinical Trials Transformation Initiative quality-by-design recommendations and principles should enable organizations to prioritize the most critical determinants of a trial's quality, identify non-essential activities that can be eliminated to streamline trial conduct and oversight, and formulate appropriate plans to define, avoid, mitigate, monitor, and address important errors.


Cholesterol, diastolic blood pressure, and stroke: 13,000 strokes in 450,000 people in 45 prospective cohorts. Prospective studies collaboration.
Individual studies of stroke have not clearly answered two questions: on the relation, if any, between total blood cholesterol and stroke; and on how the strength of the relation between diastolic blood pressure and stroke varies with age. The associations of blood cholesterol and diastolic blood pressure with subsequent stroke rates were investigated by review of 45 prospective observational cohorts involving 450,000 individuals with 5-30 years of follow-up (mean 16 years, total 7.3 million person-years of observation), during which 13,397 participants were recorded as having had a stroke. Most of these were fatal strokes in studies that recorded only mortality and not incidence, but about one-quarter were from studies that recorded both fatal and non-fatal strokes. After standardization for age, there was no association between blood cholesterol and stroke except, perhaps, in those under 45 years of age when screened. This lack of association was not influenced by adjustment for sex, diastolic blood pressure, history of coronary disease, or ethnicity (Asian or non-Asian). However, because the types of the strokes were not centrally available, the lack of any overall relation might conceal a positive association with ischaemic stroke together with a negative association with haemorrhagic stroke. When the highest and the lowest of the six blood pressure categories were compared, the difference in usual diastolic blood pressure was 27 mm Hg (102 vs 75 mm Hg), and there was a fivefold difference in stroke risk. This fivefold difference was seen both in those with a pre-existing history of coronary heart disease and in those without it. The proportional difference in stroke risk, however, was more extreme in middle than in old age. Among those aged < 45, 45-64, and 65+ when screened, the differences in the relative risks of stroke (between the highest diastolic blood pressure category and a combination of the lowest two categories) were tenfold, fivefold, and twofold, respectively. However, because the absolute stroke risks are greater in old age, the absolute differences in the annual stroke rates showed an opposite pattern, being 2, 5, and 8 per thousand, respectively. This suggests that the effects of therapeutic blood pressure reductions should be assessed separately in middle age and in old age.


Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level.
The transcription factor FOXP3 plays a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4(+)CD25(+) cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti-FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow-cytometric analysis showed that FOXP3 was expressed by the majority of CD4(+)CD25(high) T cells in peripheral blood. By contrast, less than half of the CD4(+)CD25(int) population were FOXP3(+), providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4(+)CD25(high) pool. Although FOXP3 expression was primarily restricted to CD4(+)CD25(+) cells, it was induced following activation of both CD4(+) and CD8(+) T cell clones. These findings indicate that the frequency of FOXP3(+) cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4(+) and CD8(+) T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.


Mental training enhances attentional stability: neural and behavioral evidence.
The capacity to stabilize the content of attention over time varies among individuals, and its impairment is a hallmark of several mental illnesses. Impairments in sustained attention in patients with attention disorders have been associated with increased trial-to-trial variability in reaction time and event-related potential deficits during attention tasks. At present, it is unclear whether the ability to sustain attention and its underlying brain circuitry are transformable through training. Here, we show, with dichotic listening task performance and electroencephalography, that training attention, as cultivated by meditation, can improve the ability to sustain attention. Three months of intensive meditation training reduced variability in attentional processing of target tones, as indicated by both enhanced theta-band phase consistency of oscillatory neural responses over anterior brain areas and reduced reaction time variability. Furthermore, those individuals who showed the greatest increase in neural response consistency showed the largest decrease in behavioral response variability. Notably, we also observed reduced variability in neural processing, in particular in low-frequency bands, regardless of whether the deviant tone was attended or unattended. Focused attention meditation may thus affect both distracter and target processing, perhaps by enhancing entrainment of neuronal oscillations to sensory input rhythms, a mechanism important for controlling the content of attention. These novel findings highlight the mechanisms underlying focused attention meditation and support the notion that mental training can significantly affect attention and brain function.


Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy.
An enigmatic feature of Lyme disease is the slow resolution of musculoskeletal symptoms that can continue after treatment, with some patients developing an inflammatory arthritis that becomes refractory to antibiotic therapy. Using intravital microscopy and the mouse model of Lyme borreliosis, we observed that Borrelia burgdorferi antigens, but not infectious spirochetes, can remain adjacent to cartilage for extended periods after antibiotic treatment. B. burgdorferi was not recovered by culture or xenodiagnosis with ticks after antibiotic treatment of WT mice and all but one of the immunodeficient mice with heightened pathogen burden due to impaired TLR responsiveness. Amorphous GFP+ deposits were visualized by intravital microscopy in the entheses of antibiotic-treated mice infected with GFP-expressing spirochetes and on the ear cartilage surface in sites where immunofluorescence staining detected spirochete antigens. Naive mice were not infected by tissue transplants from antibiotic-treated mice even though transplants contained spirochete DNA. Tissue homogenates from antibiotic-treated mice induced IgG reactive with B. burgdorferi antigens after immunization of naive mice and stimulated TNF-α production from macrophages in vitro. This is the first direct demonstration that inflammatory B. burgdorferi components can persist near cartilaginous tissue after treatment for Lyme disease. We propose that these deposits could contribute to the development of antibiotic-refractory Lyme arthritis.


Correction of ocular and atmospheric wavefronts: a comparison of the performance of various deformable mirrors.
The main applications of adaptive optics are the correction of the effects of atmospheric turbulence on ground-based telescopes and the correction of ocular aberrations in retinal imaging and visual simulation. The requirements for the wavefront corrector, usually a deformable mirror, will depend on the statistics of the aberrations to be corrected; here we compare the spatial statistics of wavefront aberrations expected in these two applications. We also use measured influence functions and numerical simulations to compare the performance of eight commercially available deformable mirrors for these tasks. The performance is studied as a function of the size of the optical pupil relative to the actuated area of the mirrors and as a function of the number of modes corrected. In the ocular case it is found that, with the exception of segmented mirrors, the performance is greatly enhanced by having a ring of actuators outside the optical pupil, as this improves the correction of the pupil edge. The effect is much smaller in the case of Kolmogorov wavefronts. It is also found that a high Strehl ratio can be obtained in the ocular case with a relatively low number of actuators if the stroke is sufficient. Increasing the number of actuators has more importance in the Kolmogorov case, even for the relatively weak turbulence considered here.


Genetic architecture of white matter hyperintensities differs in hypertensive and nonhypertensive ischemic stroke.
Epidemiological studies suggest that white matter hyperintensities (WMH) are extremely heritable, but the underlying genetic variants are largely unknown. Pathophysiological heterogeneity is known to reduce the power of genome-wide association studies (GWAS). Hypertensive and nonhypertensive individuals with WMH might have different underlying pathologies. We used GWAS data to calculate the variance in WMH volume (WMHV) explained by common single nucleotide polymorphisms (SNPs) as a measure of heritability (SNP heritability [HSNP]) and tested the hypothesis that WMH heritability differs between hypertensive and nonhypertensive individuals. WMHV was measured on MRI in the stroke-free cerebral hemisphere of 2336 ischemic stroke cases with GWAS data. After adjustment for age and intracranial volume, we determined which cardiovascular risk factors were independent predictors of WMHV. Using the genome-wide complex trait analysis tool to estimate HSNP for WMHV overall and within subgroups stratified by risk factors found to be significant in multivariate analyses. A significant proportion of the variance of WMHV was attributable to common SNPs after adjustment for significant risk factors (HSNP=0.23; P=0.0026). HSNP estimates were higher among hypertensive individuals (HSNP=0.45; P=7.99×10(-5)); this increase was greater than expected by chance (P=0.012). In contrast, estimates were lower, and nonsignificant, in nonhypertensive individuals (HSNP=0.13; P=0.13). A quarter of variance is attributable to common SNPs, but this estimate was greater in hypertensive individuals. These findings suggest that the genetic architecture of WMH in ischemic stroke differs between hypertensives and nonhypertensives. Future WMHV GWAS studies may gain power by accounting for this interaction.


[Epidemiology of major depressive episodes among Chinese adults aged 30-79 years: data from the China Kadoorie Biobank].
To examine the socio-demographic patterns on 12-month major depressive episodes (MDE) among Chinese adults aged 30-79 years. Socio-demographic variables from half a million Chinese people aged 30-79 years in the China Kadoorie Biobank study during 2004-2008, were collected. For those showing signs of depression, an additional World Health Organization Composite International Diagnostic Interview-Short Form (CIDI-SF) was face to face administrated, to assess the MDE according to Diagnostic and Statistical Manual of Mental Disorder (DSM)- IV criteria. Chi squared and non-parametric tests were used to compare the differences between socio-demographic categories, depressive symptoms and help-seeking behaviors. Associations with MDE for socio-demographic variables were examined by logistic models. A total of 3 281(6.40‰) studied subjects showed an MDE in the preceding 12 months. Gender, marital status, occupation and household income were correlated with MDE. Somatization was common in Chinese MDE patients while symptoms as weight change (99.91%), sleeping disorder (83.60%) and feeling tired (81.59%), stood for the top 3. As for each episode, the proportions and the total number of depressive symptoms appeared higher among females (P < 0.05). One third of the patients did not seek for any social support or psychotherapy with only 8.99% had ever taken antidepressant drugs. Rates of depression varied significantly between social groups in Chinese adults aged 30-79. In this study, only 65.19% of the MDE patients ever has sought help or received treatment.


Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.
Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated). We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.


Causes of metabolic acidosis in canine hemorrhagic shock: role of unmeasured ions.
Metabolic acidosis during hemorrhagic shock is common and conventionally considered to be due to hyperlactatemia. There is increasing awareness, however, that other nonlactate, unmeasured anions contribute to this type of acidosis. Eleven anesthetized dogs were hemorrhaged to a mean arterial pressure of 45 mm Hg and were kept at this level until a metabolic oxygen debt of 120 mLO2/kg body weight had evolved. Blood pH, partial pressure of carbon dioxide, and concentrations of sodium, potassium, magnesium, calcium, chloride, lactate, albumin, and phosphate were measured at baseline, in shock, and during 3 hours post-therapy. Strong ion difference and the amount of weak plasma acid were calculated. To detect the presence of unmeasured anions, anion gap and strong ion gap were determined. Capillary electrophoresis was used to identify potential contributors to unmeasured anions. During induction of shock, pH decreased significantly from 7.41 to 7.19. The transient increase in lactate concentration from 1.5 to 5.5 mEq/L during shock was not sufficient to explain the transient increases in anion gap (+11.0 mEq/L) and strong ion gap (+7.1 mEq/L), suggesting that substantial amounts of unmeasured anions must have been generated. Capillary electrophoresis revealed increases in serum concentration of acetate (2.2 mEq/L), citrate (2.2 mEq/L), alpha-ketoglutarate (35.3 microEq/L), fumarate (6.2 microEq/L), sulfate (0.1 mEq/L), and urate (55.9 microEq/L) after shock induction. Large amounts of unmeasured anions were generated after hemorrhage in this highly standardized model of hemorrhagic shock. Capillary electrophoresis suggested that the hitherto unmeasured anions citrate and acetate, but not sulfate, contributed significantly to the changes in strong ion gap associated with induction of shock.


Differential effects of cyclosporin A and tacrolimus on magnesium influx in Caco2 cells.
Hypomagnesemia with urinary magnesium (Mg) wasting is a well acknowledged side effect of cyclosporin A (CsA) and tacrolimus (FK506) treatments. TRPM6, TRPM7 and MagT1 are involved in the active transcellular Mg transport processes in intestine and kidney. Since Mg homeostasis is tightly controlled by the dynamic action of intestinal absorption of dietary Mg and renal excretion of Mg, we investigated whether CsA and FK506 in commercially available solutions for clinical use decrease the expression and the function of TRPM6, TRPM7 or MagT1 in the intestinal epithelial cell line Caco2. Changes of intracellular free Mg concentrations were measured by Mag-fura-2 imaging in Mg-free medium after the addition of 1 mM MgCl2. TRPM6, TRPM7 and MagT1 were evidenced in cells by immunofluorescence. Proteins and mRNAs were quantified after 18 hours of treatment with CsA or FK506 by western-blot and real-time RT-PCR analyses, respectively. TRPM6 and MagT1 were evidenced on all cell membranes, TRPM7 only on the inner membranes. CsA was responsible for a profound decrease in Mg2+ influx in intestinal epithelial cells, which may result in a decrease of intestinal Mg absorption, whereas FK506 was responsible for a marked increase in Mg2+ influx. Neither CsA nor FK506 altered TRPM6, TRPM7 or MagT1 mRNA levels or MagT1 protein level. In Caco2 cells, Mg2+ influx was inhibited by CsA solutions whereas enhanced by FK506 solutions, without alteration of MagT1, TRPM6 and TRPM7 expression, leading to the conclusion that CsA and FK506 have opposite effects in the functional activity of the Mg transporters herein examined. In clinical use, FK506 should be preferred for patients at risk for hypomagnesemia.


The C-terminus of p63 contains multiple regulatory elements with different functions.
The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ∼13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63.


Polymorphisms in the catechol-O-methyltransferase (COMT) gene influence plasma total homocysteine levels.
Elevated plasma total homocysteine (tHcy) is a risk factor for various disorders. We investigated whether functional polymorphisms in catechol-O-methyltransferase (COMT) influence tHcy, since COMT activity produces S-adenosylhomocysteine (SAH), a homocysteine precursor. We hypothesized that high activity COMT variants would be associated with high tHcy, since they presumably result in increased formation of SAH. We genotyped 780 community-dwelling elderly individuals for functional COMT (Val(158)Met and A(-287)G) and methylenetetrahydrofolate reductase (MTHFR; C(677)T) polymorphisms, and measured plasma tHcy. As predicted, COMT Val(158) carriers had significantly higher tHcy than Met(158) homozygotes. The effect was limited to individuals homozygous for the MTHFR T(677) allele. In addition, individuals homozygous for the COMT G(-287) allele tended to have lower tHcy levels. High activity variants of COMT interact with the low activity variant of MTHFR to increase tHcy levels. The effect on tHcy may contribute to the reported associations of COMT genotype with psychiatric and neurobiological phenotypes. The results also indicate that COMT activity may influence a broader range of biochemical pathways than hitherto appreciated.


Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection.
Dengue virus infection is an increasingly important tropical disease, causing 100 million cases each year. Symptoms range from mild febrile illness to severe hemorrhagic fever. The pathogenesis is incompletely understood, but immunopathology is thought to play a part, with antibody-dependent enhancement and massive immune activation of T cells and monocytes/macrophages leading to a disproportionate production of proinflammatory cytokines. We sought to investigate whether a defective population of regulatory T cells (T reg cells) could be contributing to immunopathology in severe dengue disease. CD4(+)CD25(high)FoxP3(+) T reg cells of patients with acute dengue infection of different severities showed a conventional phenotype. Unexpectedly, their capacity to suppress T cell proliferation and to secrete interleukin-10 was not altered. Moreover, T reg cells suppressed the production of vasoactive cytokines after dengue-specific stimulation. Furthermore, T reg cell frequencies and also T reg cell/effector T cell ratios were increased in patients with acute infection. A strong indication that a relative rise of T reg cell/effector T cell ratios is beneficial for disease outcome comes from patients with mild disease in which this ratio is significantly increased (P < 0.0001) in contrast to severe cases (P = 0.2145). We conclude that although T reg cells expand and function normally in acute dengue infection, their relative frequencies are insufficient to control the immunopathology of severe disease.


Healthcare and social services resource use and costs of self-harm patients.
Patients who have self-harmed have increased morbidity across a wide range of health outcomes, but there is no evidence on their pattern of health and social service use, and its relationship with repetition of self-harm. Previous studies have shown that resource use and costs in the short-term hospital management of self-harm is associated with certain patient and service characteristics but their impact in the longer term has not been demonstrated. The aim of this study is to test the association between changing levels of costs of health and social care with further episodes of self-harm and to identify the clinical and social factors associated with this. This was a cost-analysis incidence study of a sample of patients from a cohort of self-harm patients who remained within one region over the course of their follow-up. Resource use was retrospectively observed from their first episode of self-harm (dating back on some occasions to the 1970's), and costs applied. Panel data analyses were used to identify factors associated with observed costs over time. Patients with five or more episodes of self-harm had the highest levels of resource costs. Health and social care costs reduced with time from last episode of self-harm. In the year following the first episode of self-harm, psychiatric care accounted for 69% and psychotropic drug prescriptions 1% of the mean resource costs. The management of self-harm occurs within a complex system of health and social care. Major self-harm repeaters place the greatest cost burden on the system. Better understanding of the impact of risk assessment models and consequent service provision on clinical outcome may help in the design of effective services for this patient group.


The structure of an RNAi polymerase links RNA silencing and transcription.
RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-A resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world.


Activation of Myenteric Glia during Acute Inflammation In Vitro and In Vivo.
Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network. In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response. Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine.


The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus.
The cell wall synthesis pathway producing peptidoglycan is a highly coordinated and tightly regulated process. Although the major components of bacterial cell walls have been known for decades, the complex regulatory network controlling peptidoglycan synthesis and many details of the cell division machinery are not well understood. The eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp play an important role in cell wall biosynthesis and drug resistance in S. aureus. We show that stp deletion has a pronounced impact on cell wall synthesis. Deletion of stp leads to a thicker cell wall and decreases susceptibility to lysostaphin. Stationary phase Δstp cells accumulate peptidoglycan precursors and incorporate higher amounts of incomplete muropeptides with non-glycine, monoglycine and monoalanine interpeptide bridges into the cell wall. In line with this cell wall phenotype, we demonstrate that the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. Mass spectrometric analyses identify Thr32, Thr36 and Ser415 as phosphoacceptors. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Moreover, Stk interacts with FemA and FemB, but is unable to phosphorylate them. Our data indicate that Stk and Stp modulate cell wall synthesis and cell division at several levels.


Improved imputation quality of low-frequency and rare variants in European samples using the 'Genome of The Netherlands'.
Although genome-wide association studies (GWAS) have identified many common variants associated with complex traits, low-frequency and rare variants have not been interrogated in a comprehensive manner. Imputation from dense reference panels, such as the 1000 Genomes Project (1000G), enables testing of ungenotyped variants for association. Here we present the results of imputation using a large, new population-specific panel: the Genome of The Netherlands (GoNL). We benchmarked the performance of the 1000G and GoNL reference sets by comparing imputation genotypes with 'true' genotypes typed on ImmunoChip in three European populations (Dutch, British, and Italian). GoNL showed significant improvement in the imputation quality for rare variants (MAF 0.05-0.5%) compared with 1000G. In Dutch samples, the mean observed Pearson correlation, r(2), increased from 0.61 to 0.71. We also saw improved imputation accuracy for other European populations (in the British samples, r(2) improved from 0.58 to 0.65, and in the Italians from 0.43 to 0.47). A combined reference set comprising 1000G and GoNL improved the imputation of rare variants even further. The Italian samples benefitted the most from this combined reference (the mean r(2) increased from 0.47 to 0.50). We conclude that the creation of a large population-specific reference is advantageous for imputing rare variants and that a combined reference panel across multiple populations yields the best imputation results.


Preeclampsia, prematurity and cardiovascular health in adult life.
Investigations into how perinatal growth and intrauterine environment may 'programme' risk of later cardiovascular disease have been ongoing for over two decades. One of the more recent outcomes of these studies is the observation that certain pregnancy-related conditions, such as preterm birth, have an unusually large impact on the long-term cardiovascular health of the offspring. In the present paper, we review the current literature of how preterm birth affects the long-term cardiovascular structure and function of the offspring, considering three major areas of investigation: firstly, outlining the long-term cardiovascular phenotypic changes in preterm-born individuals; secondly, investigating factors related to preterm birth that may be modifying cardiovascular phenotype, such as preeclampsia, perinatal interventions, and physiological disturbances; and thirdly, the expected clinical relevance of these cardiovascular changes. This review discusses the importance of continued research focused on the mechanistic understanding of these cardiovascular alterations in order to develop specific primary prevention strategies.


Pyruvate dehydrogenase as a therapeutic target for obesity cardiomyopathy.
Obesity cardiomyopathy is a major public health problem with few specific therapeutic options. Abnormal cardiac substrate metabolism with reduced pyruvate dehydrogenase (PDH) activity is associated with energetic and functional cardiac impairment and may be a therapeutic target. This review summarizes the changes to cardiac substrate and high energy phosphorus metabolism that occur in obesity and describes the links between abnormal metabolism and impairment of cardiac function. The available evidence for the currently available pharmacological options for selective metabolic therapy in obesity cardiomyopathy is reviewed. Pharmacological restoration of PDH activity is in general associated with favourable effects upon cardiac substrate metabolism and function in both animal models and small scale human studies, supporting a potential role as a therapeutic target.


Use of whole embryo culture for studying heart development.
Congenital heart defects occur in approximately 1% of newborns and are a major cause of morbidity and mortality in infants and children. Many adult cardiac diseases also have developmental basis, such as heart valve malformations, among others. Therefore, dissecting the developmental and molecular mechanisms underlying such defects in embryos is of great importance in prevention and developing therapeutics for heart diseases that manifest in infants or later in adults. Whole embryo culture is a valuable tool to study cardiac development in midgestation embryos, in which ventricular chambers are specified and expand, and the myocardium and endocardium interact to form various cardiac structures including heart valves and trabecular myocardium (Cell 118: 649-663, 2004; Dev Cell 14: 298-311, 2008). This technique is essentially growing a midgestation embryo ex utero in a test tube. One of the strengths of embryo culture is that it allows an investigator to easily manipulate or add drugs/chemicals directly to the embryos to test specific hypotheses in situations that are otherwise very difficult to perform for embryos in utero. For instance, embryo culture permits pharmacological rescue experiments to be performed in place of genetic rescue experiments which may require generation of specific mouse strains and crosses. Furthermore, because embryos are grown externally, drugs are directly acting on the cultured embryos rather than being degraded through maternal circulation or excluded from the embryos by the placenta. Drug dosage and kinetics are therefore easier to control with embryo culture. Conversely, drugs that compromise the placental function and are thus unusable for in utero experiments are applicable in cultured embryos since placental function is not required in whole embryo culture. The applications of whole embryo culture in the studies of molecular pathways involved in heart valve formation, myocardial growth, differentiation, and morphogenesis are demonstrated previously (Cell 118: 649-663, 2004; Dev Cell 14: 298-311, 2008; Nature 446: 62-67, 2010). Here we describe a method of embryo culture in a common laboratory setting without using special equipments.


Diabetic ketoacidosis producing extreme hyperkalemia in a patient with type 1 diabetes on hemodialysis.
Diabetic ketoacidosis (DKA) is a critical complication of type 1 diabetes associated with water and electrolyte disorders. Here, we report a case of DKA with extreme hyperkalemia (9.0 mEq/L) in a patient with type 1 diabetes on hemodialysis. He had a left frontal cerebral infarction resulting in inability to manage his continuous subcutaneous insulin infusion pump. Electrocardiography showed typical changes of hyperkalemia, including absent P waves, prolonged QRS interval and tented T waves. There was no evidence of total body water deficit. After starting insulin and rapid hemodialysis, the serum potassium level was normalized. Although DKA may present with hypokalemia, rapid hemodialysis may be necessary to resolve severe hyperkalemia in a patient with renal failure. Patients with type 1 diabetes on hemodialysis may develop ketoacidosis because of discontinuation of insulin treatment.Patients on hemodialysis who develop ketoacidosis may have hyperkalemia because of anuria.Absolute insulin deficit alters potassium distribution between the intracellular and extracellular space, and anuria abolishes urinary excretion of potassium.Rapid hemodialysis along with intensive insulin therapy can improve hyperkalemia, while fluid infusions may worsen heart failure in patients with ketoacidosis who routinely require hemodialysis.


p53 gene targeting by homologous recombination in fish ES cells.
Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes). Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.


Moderate alcohol consumption as risk factor for adverse brain outcomes and cognitive decline: longitudinal cohort study.
Objectives To investigate whether moderate alcohol consumption has a favourable or adverse association or no association with brain structure and function.Design Observational cohort study with weekly alcohol intake and cognitive performance measured repeatedly over 30 years (1985-2015). Multimodal magnetic resonance imaging (MRI) was performed at study endpoint (2012-15).Setting Community dwelling adults enrolled in the Whitehall II cohort based in the UK (the Whitehall II imaging substudy).Participants 550 men and women with mean age 43.0 (SD 5.4) at study baseline, none were "alcohol dependent" according to the CAGE screening questionnaire, and all safe to undergo MRI of the brain at follow-up. Twenty three were excluded because of incomplete or poor quality imaging data or gross structural abnormality (such as a brain cyst) or incomplete alcohol use, sociodemographic, health, or cognitive data.Main outcome measures Structural brain measures included hippocampal atrophy, grey matter density, and white matter microstructure. Functional measures included cognitive decline over the study and cross sectional cognitive performance at the time of scanning.Results Higher alcohol consumption over the 30 year follow-up was associated with increased odds of hippocampal atrophy in a dose dependent fashion. While those consuming over 30 units a week were at the highest risk compared with abstainers (odds ratio 5.8, 95% confidence interval 1.8 to 18.6; P≤0.001), even those drinking moderately (14-21 units/week) had three times the odds of right sided hippocampal atrophy (3.4, 1.4 to 8.1; P=0.007). There was no protective effect of light drinking (1-<7 units/week) over abstinence. Higher alcohol use was also associated with differences in corpus callosum microstructure and faster decline in lexical fluency. No association was found with cross sectional cognitive performance or longitudinal changes in semantic fluency or word recall.Conclusions Alcohol consumption, even at moderate levels, is associated with adverse brain outcomes including hippocampal atrophy. These results support the recent reduction in alcohol guidance in the UK and question the current limits recommended in the US.


Improving poisoning diagnosis and surveillance of street pesticides.
An effective surveillance system is required to reduce pesticide exposures and poisonings, especially from street pesticides (illegal, unlabelled, and decanted agricultural pesticides used predominately for urban household purposes). Poisoning from any pesticide class, not only organophosphates, constitutes a medically notifiable condition in South Africa. Current practice, however, is to report only organophosphate cases, resulting in severe under-reporting. The lack of data concerning the link between poisonings and street pesticides has led to the mistaken assumption that urban populations are not at risk from significant pesticide exposures and poisonings. Without accurate statistics, healthcare professionals and policy makers are unaware of the contribution of street pesticide poisonings to the overall health burden. Accurate diagnosis is a prerequisite for notification and subsequent surveillance. An algorithm has been developed to enable healthcare professionals to improve the diagnosis and notification of pesticide poisonings.