ß-importin Tnpo-SR promotes germline stem cell maintenance and oocyte differentiation in female Drosophila.

Germ cell development requires interplay between factors that balance cell fate and division. Early in their development, germ cells in many organisms divide mitotically with incomplete cytokinesis. Key regulatory events then lead to the specification of mature gametes, marked by the switch to a meiotic cell cycle program. Though the regulation of germ cell proliferation and meiosis are well understood, how these events are coordinated during development remains incompletely described. Originally characterized in their role as nucleo-cytoplasmic shuttling proteins, ß-importins exhibit diverse functions during male and female gametogenesis. Here, we describe novel, distinct roles for the ß-importin, Transportin-Serine/Arginine rich (Tnpo-SR), as a regulator of the mitosis to meiosis transition in the Drosophila ovary. We find that Tnpo-SR is necessary for germline stem cell (GSC) establishment and self-renewal, likely by controlling the response of GSCs to bone morphogenetic proteins. Depletion of Tnpo-SR results in germ cell counting defects and loss of oocyte identity. We show that in the absence of Tnpo-SR, proteins typically suppressed in germ cells when they exit mitosis fail to be down-regulated, and oocyte-specific factors fail to accumulate. Together, these findings provide new insight into the balance between germ cell division and differentiation and identify novel roles for ß-importins in germ cell development.

Utilizing the FLP-Out System for Clonal RNAi Analysis in the Adult Drosophila Ovary.

The ability to conduct spatially controlled RNA interference (RNAi) for gene knockdown using the UAS/Gal4 system is among the most appealing techniques available for analysis of gene function in the Drosophila ovary. While gene knockdown experiments in somatic cells in the developing organism (i.e., embryos and larvae) are effectively and commonly performed, the use of RNAi in adult ovarian cells can be hampered by the unintended deleterious effects of Gal4 expression in "off-target" developing tissues. Mosaic analysis overcomes these problems by imparting temporal and spatial control over gene manipulation, providing a useful tool to compare manipulated cells with wild-type cells in the same tissue. Here, we provide a method to utilize the UAS/Gal4 system in combination with the Flippase (FLP)-Flippase Recognition Target (FRT) system to generate positively labeled "FLP-Out" clones expressing a chosen RNAi in both the germline and the soma in the Drosophila ovary. This protocol outlines each step of the generation of clones and the selection of appropriate fly stocks and reagents, providing a guide to this powerful tool in the Drosophila genetic toolbox. These techniques allow for RNAi analysis within a specific cell type, providing an opportunity to study a variety of unique aspects of cell function that would not be possible in more traditional RNAi-based experiments.

Advances in medical therapy for weight loss and the weight-centric management of type 2 diabetes mellitus.

Overweight and obesity are now recognized as leading causes of diseases such as type 2 diabetes, hypertension, hyperlipidemia, and ultimately, cardiovascular disease. Despite the serious consequences, roughly two thirds of Americans are presently classified as overweight, and about one third are classified as obese. Weight loss via lifestyle modification and pharmacotherapy can promote improvement in many of these obesity-related conditions. This review addresses recent advances in pharmacotherapy for the management of obesity and obesity-related co-morbidities, with a focus on the management of obesity specifically in individuals with type 2 diabetes. Emphasis is also placed on a proposed paradigm shift from the glucose-centric to the weight-centric management of type 2 diabetes mellitus.

Emerging pharmacotherapy for obesity.

INTRODUCTION: Obesity is a rapidly increasing global health problem. The rates of obesity have tripled over the past three decades and are predicted to rise even further. The need for safe and effective therapies is great and has gone unmet. AREAS COVERED: This review will summarize the emerging pharmacological treatments for obesity. The research discussed includes Phase III and early-phase clinical trials as well as recently published data regarding emerging treatment therapies. EXPERT OPINION: The new generation of anti-obesity drugs offers hope in the management of obesity, but no single agent is likely to be a panacea. Treatment of obesity will likely use multiple medications to achieve the desired result. Additionally, in light of the challenges faced by drug companies in order to gain FDA approval for an obesity therapy, the future approach may be to seek approval for obesity-related co-morbidities. This approach would enhance the ability of physicians to practice 'weight-centric' disease management.

Obesity: Pharmacotherapy.

Pharmacotherapy, adjunctively with lifestyle interventions, is an option for any patient diagnosed with obesity (ie, body mass index [BMI] of 30 kg/m2 or greater) or with a BMI of 27 kg/m2 or greater and at least one coexisting condition, including type 2 diabetes, hypertension, hyperlipidemia, and sleep apnea. If the appropriate criteria are met, pharmacotherapy should be initiated for patients with overweight or obesity if lifestyle modification does not produce adequate weight loss. Lifestyle modifications should be continued and emphasized throughout treatment because it has been shown that adjunctive pharmacotherapy produces greater weight loss and weight loss maintenance than lifestyle interventions alone. Currently, five drugs are approved for weight management in adults: phentermine, orlistat, phentermine-topiramate, bupropion-naltrexone, and liraglutide. Certain drugs are approved for short-term management while others are approved for long-term management. Drug therapy should be customized to the individual patient, depending on needs, contraindications, and cost. Benefits of these drugs should be assessed regularly and a different drug should be considered if at least 5% of body weight is not lost by 3 months of therapy.

Engineered biosynthesis of nonribosomal lipopeptides with modified fatty acid side chains.

The biological properties of the calcium-dependent antibiotics (CDAs), daptomycin and related nonribosomal lipopeptides, depend to a large extent on the nature of the N-terminal fatty acid moiety. It is suggested that the chain length of the unusually short (C6) 2,3-epoxyhexanoyl fatty acid moiety of CDA is determined by the specificity of the KAS-II enzyme encoded by fabF3 in the CDA biosynthetic gene cluster. Indeed, deletion of the downstream gene hxcO results in three new lipopeptides, all of which possess hexanoyl side chains (hCDAs). This confirms that HxcO functions as a hexanoyl-CoA or -ACP oxidase. The absence of additional CDA products with longer fatty acid groups further suggests that the CDA lipid chain is biosynthesized on a single ACP and is then transferred directly from this ACP to the first CDA peptide synthetase (CdaPS1). Interestingly, the hexanoyl-containing CDAs retain antibiotic activity. To further modulate the biological properties of CDA by introducing alternative fatty acid groups, a mutasynthesis approach was developed. This involved mutating the key active site Ser residue of the CdaPS1, module 1 PCP domain to Ala, which prevents subsequent phosphopantetheinylation. In the absence of the natural module 1 PCP tethered intermediate, it is possible to effect incorporation of different N-acyl-L-serinyl N-acetylcysteamine (NAC) thioester analogues, leading to CDA products with pentanoyl as well as hexanoyl side chains.

Precursor-directed biosynthesis of nonribosomal lipopeptides with modified glutamate residues.

Precursor-directed biosynthesis of calcium dependent antibiotics (CDAs) with modified 3-trifluoromethyl and 3-ethyl glutamate residues was achieved by feeding synthetic glutamate analogues to a mutant strain of Streptomyces coelicolor impaired in the biosynthesis of the natural precursor (2S,3R)-3-methyl glutamic acid.

Statistical methods used in the public health literature and implications for training of public health professionals.

Statistical literacy and knowledge is needed to read and understand the public health literature. The purpose of this study was to quantify basic and advanced statistical methods used in public health research. We randomly sampled 216 published articles from seven top tier general public health journals. Studies were reviewed by two readers and a standardized data collection form completed for each article. Data were analyzed with descriptive statistics and frequency distributions. Results were summarized for statistical methods used in the literature, including descriptive and inferential statistics, modeling, advanced statistical techniques, and statistical software used. Approximately 81.9% of articles reported an observational study design and 93.1% of articles were substantively focused. Descriptive statistics in table or graphical form were reported in more than 95% of the articles, and statistical inference reported in more than 76% of the studies reviewed. These results reveal the types of statistical methods currently used in the public health literature. Although this study did not obtain information on what should be taught, information on statistical methods being used is useful for curriculum development in graduate health sciences education, as well as making informed decisions about continuing education for public health professionals.

Phentermine plus topiramate for the treatment of obesity.

A novel combination therapy is currently being investigated for the treatment of obesity. Phentermine plus topiramate has shown efficacy in both Phase II and III trials as compared with either drug used alone. With average 2-year weight loss as high as 10.5% and reduction of up to 76% in the incidence of progression of Type II diabetes, combination phentermine plus topiramate may provide significant benefit in conjunction with behavioral modification in the management of obesity. However, as with any new pharmacologic treatment option, the risk/benefit profile needs to be carefully assessed.

Evidence from individual inference for high-dimensional coexistence: long-term experiments on recruitment response.

BACKGROUND: For competing species to coexist, individuals must compete more with others of the same species than with those of other species. Ecologists search for tradeoffs in how species might partition the environment. The negative correlations among competing species that would be indicative of tradeoffs are rarely observed. A recent analysis showed that evidence for partitioning the environment is available when responses are disaggregated to the individual scale, in terms of the covariance structure of responses to environmental variation. That study did not relate that variation to the variables to which individuals were responding. To understand how this pattern of variation is related to niche variables, we analyzed responses to canopy gaps, long viewed as a key variable responsible for species coexistence. METHODOLOGY/PRINCIPAL FINDINGS: A longitudinal intervention analysis of individual responses to experimental canopy gaps with 12 yr of pre-treatment and 8 yr post-treatment responses showed that species-level responses are positively correlated--species that grow fast on average in the understory also grow fast on average in response to gap formation. In other words, there is no tradeoff. However, the joint distribution of individual responses to understory and gap showed a negative correlation--species having individuals that respond most to gaps when previously growing slowly also have individuals that respond least to gaps when previously growing rapidly (e.g., Morus rubra), and vice versa (e.g., Quercus prinus). CONCLUSIONS/SIGNIFICANCE: Because competition occurs at the individual scale, not the species scale, aggregated species-level parameters and correlations hide the species-level differences needed for coexistence. By disaggregating models to the scale at which the interaction occurs we show that individual variation provides insight for species differences.

A prospective analysis of the association between cardiovascular disease and depression in middle-aged women.

OBJECTIVE: Experimental and clinical data demonstrate a close association between depression and coronary heart disease (CHD). Because no simple depression instrument for use by general practitioners has been shown to predict CHD, the objective of this study was to evaluate whether such a questionnaire could predict CHD. METHODS: The prevalence of CHD and CHD risk factors was assessed in women with depression, measured by a validated three-question depression screening instrument. Among 1,919 participants in the breast arterial calcification and CHD 5-year prospective study, 1,454 women (75.8%) completed a baseline depression inventory. RESULTS: Among the 1,454 women, 72.2% were postmenopausal, and the mean (SD) age at the conclusion of a 5-year prospective study was 61.3 (12.1) years. Among the women with no CHD risk factors at baseline and with one or less positive depression responses compared with those with two or more, 1.6% versus 3.8%, respectively, had at least one CHD risk factor (P = 0.004) by the 5th year of the study. In addition, 2.3% versus 6.0%, respectively, developed CHD (P = 0.005) by the 5th year of the study. Among all women with no positive depression responses compared with those with any positive depression responses, 2.1% versus 5.6%, respectively, had developed CHD by the 5th year of the study (P = 0.002). Finally, more positive depression responses were associated with a greater prevalence of CHD. CONCLUSIONS: In this prospective study, depression-detected by a validated three-item questionnaire-was able to predict those women more likely to develop CHD.

An asparagine oxygenase (AsnO) and a 3-hydroxyasparaginyl phosphotransferase (HasP) are involved in the biosynthesis of calcium-dependent lipopeptide antibiotics.

Nonribosomal peptides contain a wide range of unusual non-proteinogenic amino acid residues. As a result, these complex natural products are amongst the most structurally diverse secondary metabolites in nature, and possess a broad spectrum of biological activities. beta-Hydroxylation of amino acid precursors or peptidyl residues and their subsequent processing by downstream tailoring enzymes are some of the most common themes in the biosynthetic diversification of these therapeutically important peptides. Identification and characterization of the biosynthetic intermediates and enzymes involved in these processes are thus pivotal in understanding nonribosomal peptide assembly and modification. To this end, the putative asparaginyl oxygenase- and 3-hydroxyasparaginyl phosphotransferase-encoding genes hasP and asnO were separately deleted from the calcium-dependent antibiotic (CDA) biosynthetic gene cluster of Streptomyces coelicolor. Whilst the parent strains produce a number of 3-hydroxyasparagine- and 3-phosphohydroxyasparagine-containing CDAs, the DeltahasP mutants produce exclusively non-phosphorylated CDAs. On the other hand, DeltaasnO mutants produce several new Asn-containing CDAs not present in the wild-type, which retain calcium-dependent antimicrobial activity. This confirms that AsnO and HasP are required for the beta-hydroxylation and phosphorylation of the Asn residue within CDA.

Biosynthesis of the (2S,3R)-3-methyl glutamate residue of nonribosomal lipopeptides.

The calcium-dependent antibiotics (CDAs) and daptomycin are therapeutically relevant nonribosomal lipopeptide antibiotics that contain penultimate C-terminal 3-methyl glutamate (3-MeGlu) residues. Comparison with synthetic standards showed that (2S,3R)-configured 3-MeGlu is present in both CDA and daptomycin. Deletion of a putative methyltransferase gene glmT from the cda biosynthetic gene cluster abolished the incorporation of 3-MeGlu and resulted in the production of Glu-containing CDA exclusively. However, the 3-MeGlu chemotype could be re-established through feeding synthetic 3-methyl-2-oxoglutarate and (2S,3R)-3-MeGlu, but not (2S,3S)-3-MeGlu. This indicates that methylation occurs before peptide assembly, and that the module 10 A-domain of the CDA peptide synthetase is specific for the (2S,3R)-stereoisomer. Further mechanistic analyses suggest that GlmT catalyzes the SAM-dependent methylation of alpha-ketoglutarate to give (3R)-methyl-2-oxoglutarate, which is transaminated to (2S,3R)-3-MeGlu. These insights will facilitate future efforts to engineer lipopeptides with modified glutamate residues, which may have improved bioactivity and/or reduced toxicity.