An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes

Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo “browning.” In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells

Regulation of glycolysis in brown adipocytes by HIF-1α

Brown adipose tissue takes up large amounts of glucose during cold exposure in mice and humans. Here we report an induction of glucose transporter 1 expression and increased expression of several glycolytic enzymes in brown adipose tissue from cold-exposed mice. Accordingly, these genes were also induced after β-adrenergic activation of cultured brown adipocytes, concomitant with accumulation of hypoxia inducible factor-1α (HIF-1α) protein levels. HIF-1α accumulation was dependent on uncoupling protein 1 and generation of mitochondrial reactive oxygen species. Expression of key glycolytic enzymes was reduced after knockdown of HIF-1α in mature brown adipocytes. Glucose consumption, lactate export and glycolytic capacity were reduced in brown adipocytes depleted of Hif-1α. Finally, we observed a decreased β-adrenergically induced oxygen consumption in Hif-1α knockdown adipocytes cultured in medium with glucose as the only exogenously added fuel. These data suggest that HIF-1α-dependent regulation of glycolysis is necessary for maximum glucose metabolism in brown adipocytes.ISSN:2045-232

LONG-TERM EFFICACY OF ELECTROCONVULSIVE THERAPY COMBINED WITH DIFFERENT ANTIPSYCHOTIC DRUGS IN PREVIOUSLY RESISTANT SHIZOPHRENIA

Background: In treatment-resistant schizophrenia a combination of ECT with antipsychotics has been reported to have superior outcomes compared to other strategies, however the results were inconsistent. We investigated the long-term effects of the combination of unilateral, non-dominant hemisphere ECT with three antipsychotics. Subjects and methods: The clinical study was a naturalistic, prospective, openlabeled, active-controlled study in adult outpatients of both genders suffering from treatment-resistant schizophrenia with a follow up of 2 years. The patients received sulpiride (n=17, 100-400mg/day, PO), risperidone (n=26, 2-8mg/day, PO) or olanzapine (n=27, 5-10mg/day, PO). Unilateral ECT was applied in 1 unit (0.5A, 0.8 mS) in six single applications, once a week and further according to the clinical need, in fortnight steps. Clinical efficacy was established using the PANSS and CGI psychometric scales. Results: According to the results, the most effective treatment mode was olanzapine plus ECT, then risperidone plus ECT, while sulpiride plus ECT had lower clinical efficacy. Olanzapine plus ECT was significantly superior in all scale scores vs sulpiride plus ECT, as well as risperidone plus ECT except for PANSS-P (t=1.85, p>0.05). During the study, 38 of 70 patients were withdrawn due to treatment failure (n=21), side effects (n=6) and non-compliance (n=11). Conclusion: The combination of novel antipsychotics and ECT can be used safely and effectively in treatment-resistant schizophrenia

Retinoic acid has different effects on UCP1 expression in mouse and human adipocytes

BACKGROUND: Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes. RESULTS: The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α. CONCLUSIONS: UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA

16p11.2 Locus modulates response to satiety before the onset of obesity

Background: The 600 kb BP4-BP5 copy number variants (CNVs) at the 16p11.2 locus have been associated with a range of neurodevelopmental conditions including autism spectrum disorders and schizophrenia. The number of genomic copies in this region is inversely correlated with body mass index (BMI): the deletion is associated with a highly penetrant form of obesity (present in 50% of carriers by the age of 7 years and in 70% of adults), and the duplication with being underweight. Mechanisms underlying this energy imbalance remain unknown. Objective: This study aims to investigate eating behavior, cognitive traits and their relationships with BMI in carriers of 16p11.2 CNVs. Methods: We assessed individuals carrying a 16p11.2 deletion or duplication and their intrafamilial controls using food-related behavior questionnaires and cognitive measures. We also compared these carriers with cohorts of individuals presenting with obesity, binge eating disorder or bulimia. Results: Response to satiety is gene dosage-dependent in pediatric CNV carriers. Altered satiety response is present in young deletion carriers before the onset of obesity. It remains altered in adolescent carriers and correlates with obesity. Adult deletion carriers exhibit eating behavior similar to that seen in a cohort of obesity without eating disorders such as bulimia or binge eating. None of the cognitive measures are associated with eating behavior or BMI. Conclusions: These findings suggest that abnormal satiety response is a strong contributor to the energy imbalance in 16p11.2 CNV carriers, and, akin to other genetic forms of obesity, altered satiety responsiveness in children precedes the increase in BMI observed later in adolescence

4 modalities of periodontal treatment compared over 5 years *

The purpose of the present study was to assess in a clinical trial over 5 years the results following 4 different modalities of periodontal therapy (pocket elimination or reduction surgery, modified Widman flap surgery, subgingival curettage, and scaling and root planing). 90 patients were treated. The treatment methods were applied on a random basis to each of the 4 quadrants of the dentition. The patients were given professional tooth cleaning and oral hygiene instructions every 3 months. Pocket depth and attachment levels were scored once a year. 72 patients completed the 5 years of observation. Both patient means for pocket depth and attachment level as well as % distribution of sites with loss of attachment ≥2 mm and ≥3 mm were compared. For 1-3 mm probing depth, scaling and root planing, as well as subgingival curettage led to significantly less attachment loss than pocket elimination and modified Widman flap surgery. For 4 6 mm pockets, scaling and root planing and curettage had better attachment results than pocket elimination surgery. For the 7-12 mm pockets, there was no statistically significant difference among the results following the various procedures.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72788/1/j.1600-051X.1987.tb02249.x.pd

The Number of Genomic Copies at the 16p11.2 Locus Modulates Language, Verbal Memory, and Inhibition.

Deletions and duplications of the 16p11.2 BP4-BP5 locus are prevalent copy number variations (CNVs), highly associated with autism spectrum disorder and schizophrenia. Beyond language and global cognition, neuropsychological assessments of these two CNVs have not yet been reported. This study investigates the relationship between the number of genomic copies at the 16p11.2 locus and cognitive domains assessed in 62 deletion carriers, 44 duplication carriers, and 71 intrafamilial control subjects. IQ is decreased in deletion and duplication carriers, but we demonstrate contrasting cognitive profiles in these reciprocal CNVs. Deletion carriers present with severe impairments of phonology and of inhibition skills beyond what is expected for their IQ level. In contrast, for verbal memory and phonology, the data may suggest that duplication carriers outperform intrafamilial control subjects with the same IQ level. This finding is reminiscent of special isolated skills as well as contrasting language performance observed in autism spectrum disorder. Some domains, such as visuospatial and working memory, are unaffected by the 16p11.2 locus beyond the effect of decreased IQ. Neuroimaging analyses reveal that measures of inhibition covary with neuroanatomic structures previously identified as sensitive to 16p11.2 CNVs. The simultaneous study of reciprocal CNVs suggests that the 16p11.2 genomic locus modulates specific cognitive skills according to the number of genomic copies. Further research is warranted to replicate these findings and elucidate the molecular mechanisms modulating these cognitive performances

BioStar: An Online Question & Answer Resource for the Bioinformatics Community

Parnell, Laurence D. et al.Although the era of big data has produced many bioinformatics tools and databases, using them effectively often requires specialized knowledge. Many groups lack bioinformatics expertise, and frequently find that software documentation is inadequate while local colleagues may be overburdened or unfamiliar with specific applications. Too often, such problems create data analysis bottlenecks that hinder the progress of biological research. In order to help address this deficiency, we present BioStar, a forum based on the Stack Exchange platform where experts and those seeking solutions to problems of computational biology exchange ideas. The main strengths of BioStar are its large and active group of knowledgeable users, rapid response times, clear organization of questions and responses that limit discussion to the topic at hand, and ranking of questions and answers that help identify their usefulness. These rankings, based on community votes, also contribute to a reputation score for each user, which serves to keep expert contributors engaged. The BioStar community has helped to answer over 2,300 questions from over 1,400 users (as of June 10, 2011), and has played a critical role in enabling and expediting many research projects. BioStar can be accessed at http://www.biostars.org/.This work was partially supported by NSF grants MCB-0618402 and CCF-0643529 (CAREER), NIH grants 1R55AI065507 – 01A2 and 1 R01 GM083113-01, NIH/NCRR grant number UL1RR033184, and FPI fellowship SAF-2007-63171/BES-2009-017731 from the Ministerio de Educación y Ciencia, Spain. These funders had no role in the design of BioStar, decision to publish, or preparation of the manuscript.Peer reviewe