Role of Fat Body Lipogenesis in Protection against the Effects of Caloric Overload in Drosophila

The Drosophila fat body is a liver- and adipose-like tissue that stores fat and serves as a detoxifying and immune responsive organ. We have previously shown that a high sugar diet leads to elevated hemolymph glucose and systemic insulin resistance in developing larvae and adults. Here, we used stable isotope tracer feeding to demonstrate that rearing larvae on high sugar diets impaired the synthesis of esterified fatty acids from dietary glucose. Fat body lipid profiling revealed changes in both carbon chain length and degree of unsaturation of fatty acid substituents, particularly in stored triglycerides. We tested the role of the fat body in larval tolerance of caloric excess. Our experiments demonstrated that lipogenesis was necessary for animals to tolerate high sugar feeding as tissue-specific loss of orthologs of carbohydrate response element-binding protein or stearoyl-CoA desaturase 1 resulted in lethality on high sugar diets. By contrast, increasing the fat content of the fat body by knockdown of king-tubby was associated with reduced hyperglycemia and improved growth and tolerance of high sugar diets. Our work supports a critical role for the fat body and the Drosophila carbohydrate response element-binding protein ortholog in metabolic homeostasis in Drosophila.     

Breeding behaviour and performance of the Knysna Warbler Bradypterus sylvaticus on the Cape Peninsula,South Africa

Three pairs of Knysna Warblers were monitored on the south-eastern slopes of Table Mountain during the 2000 breeding season. Males displayed alone on territories until the second half of August, when females arrived. Nest-building (8 days) and incubation (16 days) were undertaken entirely by the female, who was not fed on the nest by the male. Chick provisioning was done mainly by the male. Arachnids and terrestrial amphipods were the most common prey brought to chicks. The fledging period was 12 days. Modal clutch size was three eggs, and depredation rates of eggs and chicks were high. After losses, replacement clutches were laid on average 19 days later, after a new nest was built. A maximum of three clutches per pair was recorded. Of 18 eggs monitored, 28% hatched and 17% fledged, equating to a production of one fledgling per pair per year. Ten days after fledging, the entire family leaves the territory, males probably returning once young are independent. The main threats to the local populations are clearing of riparian undergrowth and management practices that impact the predators of rodents.     

C:N:P stoichiometry in Australian soils with respect to vegetation and environmental factors

Aims

We estimate organic carbon (C): total nitrogen (N): total phosphorus (P) ratios in soils under Australia’s major native vegetation groups.

Methods

We use digital datasets for climate, soils, and vegetation created for the National Land and Water Resources Audit in 2001. Analysis-of-variance is used to investigate differences in nutrient ratios between ecosystems. Linear discriminant analysis and logistic regression are used to investigate the relative importance of climatic variables and soil nutrients in vegetation patterns.

Results

We find that the N:P and C:P ratios have a greater range of values than the C:N ratio, although major vegetation groups tend to show similar trends across all three ratios. Some apparently homeostatic groupings emerge: those with very low, low, medium, or high N:P and C:P. Tussock grasslands have very low soil N, N:P, and C:P, probably due to frequent burning. Eucalypt woodlands have low soil N:P and C:P ratios, although their total P level varies. Rainforests and Melaleuca forests have medium soil N:P and C:P ratios, although their total P level is different. Heathlands, tall open eucalypt forests, and shrublands occur on soils with low levels of total P, and high N:P and C:P ratios that reflect foliar nutrient ratios and recalcitrant litter.

Conclusions

Certain plant communities have typical soil nutrient stoichiometries but there is no single Redfield-like ratio. Vegetation patterns largely reflect soil moisture but for several plant communities, eucalypt communities in particular, soil N and P (or N:P) also play a significant role. Soil N:P and the presence of Proteaceae appear indicative of nutrient constraints in ecosystems.     

RapA, Escherichia coli RNA polymerase SWI/SNF subunit-dependent polyadenylation of RNA

In this work, we describe RapA-dependent polyadenylation of model RNA substrates and endogenous, RNA polymerase-associated nucleic acid fragments. We demonstrate that the Escherichia coli RNA polymerase obtained through the classic purification procedure carries endogenous RNA oligonucleotides, which, in the presence of ATP, are polyriboadenylated in a RapA-dependent manner by an accessory poly(rA) polymerase. RNA polymerase isolated from poly(A) polymerase- (PAP-) and polynucleotide phosphorylase- (PNP-) deficient E. coli strain lacks accessory (rA)(n)-synthetic activity. Experiments with reconstituted RNA polymerase-PAP and RNA polymerase-PNP mixtures suggest that RapA enables the polyadenylation by PAP of RNA polymerase-associated RNA. Mutations disrupting RapA's ATP-hydrolytic function disrupt RapA-dependent polyadenylation, and the rapA(-)E. coli strain displays a measurable reduction in RNA polyadenylation. RapA-dependent polyadenylation can also be modulated by mutations in the section of RapA's SWI/SNF domain linked to interaction with single-stranded nucleic acid. We have developed enzymatic assays in which model, synthetic RNAs are polyriboadenylated in a RapA-dependent manner. Taken together, our results are consistent with RapA acting as an RNA polymerase-associated, ATP-dependent RNA translocase. Our work further links RapA to RNA remodeling and provides new mechanistic insights into the functional interaction between RNA polymerase and RapA.     

Multiple mechanisms of uranium immobilization by Cellulomonas sp. strain ES6

Removal of hexavalent uranium (U(VI)) from aqueous solution was studied using a Gram‐positive facultative anaerobe, Cellulomonas sp. strain ES6, under anaerobic, non‐growth conditions in bicarbonate and PIPES buffers. Inorganic phosphate was released by cells during the experiments providing ligands for formation of insoluble U(VI) phosphates. Phosphate release was most probably the result of anaerobic hydrolysis of intracellular polyphosphates accumulated by ES6 during aerobic growth. Microbial reduction of U(VI) to U(IV) was also observed. However, the relative magnitudes of U(VI) removal by abiotic (phosphate‐based) precipitation and microbial reduction depended on the buffer chemistry. In bicarbonate buffer, X‐ray absorption fine structure (XAFS) spectroscopy showed that U in the solid phase was present primarily as a non‐uraninite U(IV) phase, whereas in PIPES buffer, U precipitates consisted primarily of U(VI)‐phosphate. In both bicarbonate and PIPES buffer, net release of cellular phosphate was measured to be lower than that observed in U‐free controls suggesting simultaneous precipitation of U and PO. In PIPES, U(VI) phosphates formed a significant portion of U precipitates and mass balance estimates of U and P along with XAFS data corroborate this hypothesis. High‐resolution transmission electron microscopy (HR‐TEM) and energy dispersive X‐ray spectroscopy (EDS) of samples from PIPES treatments indeed showed both extracellular and intracellular accumulation of U solids with nanometer sized lath structures that contained U and P. In bicarbonate, however, more phosphate was removed than required to stoichiometrically balance the U(VI)/U(IV) fraction determined by XAFS, suggesting that U(IV) precipitated together with phosphate in this system. When anthraquinone‐2,6‐disulfonate (AQDS), a known electron shuttle, was added to the experimental reactors, the dominant removal mechanism in both buffers was reduction to a non‐uraninite U(IV) phase. Uranium immobilization by abiotic precipitation or microbial reduction has been extensively reported; however, the present work suggests that strain ES6 can remove U(VI) from solution simultaneously through precipitation with phosphate ligands and microbial reduction, depending on the environmental conditions. Cellulomonadaceae are environmentally relevant subsurface bacteria and here, for the first time, the presence of multiple U immobilization mechanisms within one organism is reported using Cellulomonas sp. strain ES6. Biotechnol. Bioeng. 2011;108: 264–276. © 2010 Wiley Periodicals, Inc.     

Corrections for measurement error due to delayed onset of illness for case-crossover designs

Epidemiologic studies of the short-term effects of ambient particulate matter (PM) on the risk of acute cardiovascular or cerebrovascular events often use data from administrative databases in which only the date of hospitalization is known. A common study design for analyzing such data is the case-crossover design, in which exposure at a time when a patient experiences an event is compared to exposure at times when the patient did not experience an event within a case-control paradigm. However, the time of true event onset may precede hospitalization by hours or days, which can yield attenuated effect estimates. In this article, we consider a marginal likelihood estimator, a regression calibration estimator, and a conditional score estimator, as well as parametric bootstrap versions of each, to correct for this bias. All considered approaches require validation data on the distribution of the delay times. We compare the performance of the approaches in realistic scenarios via simulation, and apply the methods to analyze data from a Boston-area study of the association between ambient air pollution and acute stroke onset. Based on both simulation and the case study, we conclude that a two-stage regression calibration estimator with a parametric bootstrap bias correction is an effective method for correcting bias in health effect estimates arising from delayed onset in a case-crossover study.     

Gene expression in the social behavior network of the wire‐tailed manakin (Pipra filicauda) brain

The vertebrate basal forebrain and midbrain contain a set of interconnected nuclei that control social behavior. Conserved anatomical structures and functions of these nuclei have now been documented among fish, amphibians, reptiles, birds and mammals, and these brain regions have come to be known as the vertebrate social behavior network (SBN). While it is known that nuclei (nodes) of the SBN are rich in steroid and neuropeptide activity linked to behavior, simultaneous variation in the expression of neuroendocrine genes among several SBN nuclei has not yet been described in detail. In this study, we use RNA‐seq to profile gene expression across seven brain regions representing five nodes of the vertebrate SBN in a passerine bird, the wire‐tailed manakin Pipra filicauda. Using weighted gene co‐expression network analysis, we reconstructed sets of coregulated genes, showing striking patterns of variation in neuroendocrine gene expression across the SBN. We describe regional variation in gene networks comprising a broad set of hormone receptors, neuropeptides, steroidogenic enzymes, catecholamines and other neuroendocrine signaling molecules. Our findings show heterogeneous patterns of brain gene expression across nodes of the avian SBN and provide a foundation for future analyses of how the regulation of gene networks may mediate social behavior. These results highlight the importance of region‐specific sampling in studies of the mechanisms of behavior.     

Somatic Mutations,Allele Loss,and DNA Methylation of the Cub and Sushi Multiple Domains 1 (CSMD1) Gene Reveals Association with Early Age of Diagnosis in Colorectal Cancer Patients

Background

The Cub and Sushi Multiple Domains 1 (CSMD1) gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients.

Methods

We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A.

Results

Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%). The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15∶1, a ratio significantly higher than the expected 2∶1 ratio (p = 0.014). This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%). Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years) than those without somatic mutations (average age, 68 years). The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%), suggesting extensive cytosine methylation predisposing to somatic mutations.

Conclusions

Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical presentation in colorectal tumors, thus further implicating CSMD1 as a tumor suppressor gene.     

Regulating the Way to Obesity: Unintended Consequences of Limiting Sugary Drink Sizes

Objectives

We examined whether a sugary drink limit would still be effective if larger-sized drinks were converted into bundles of smaller-sized drinks.

Methods

In a behavioral simulation, participants were offered varying food and drink menus. One menu offered 16 oz, 24 oz, or 32 oz drinks for sale. A second menu offered 16 oz drinks, a bundle of two 12 oz drinks, or a bundle of two 16 oz drinks. A third menu offered only 16 oz drinks for sale. The method involved repeated elicitation of choices, and the instructions did not mention a limit on drink size.

Results

Participants bought significantly more ounces of soda with bundles than with varying-sized drinks. Total business revenue was also higher when bundles rather than only small-sized drinks were sold.

Conclusions

Our research suggests that businesses have a strong incentive to offer bundles of soda when drink size is limited. Restricting larger-sized drinks may have the unintended consequence of increasing soda consumption rather than decreasing it.     

Tissue Inhibitor of Metalloproteinases-3 Peptides Inhibit Angiogenesis and Choroidal Neovascularization in Mice

Tissue inhibitors of metalloproteinases (TIMPs) while originally characterized as inhibitors of matrix metalloproteinases (MMPs) have recently been shown to have a wide range of functions that are independent of their MMP inhibitory properties. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a potent inhibitor of VEGF-mediated angiogenesis and neovascularization through its ability to block the binding of VEGF to its receptor VEGFR-2. To identify and characterize the anti-angiogenic domain of TIMP-3, structure function analyses and synthetic peptide studies were performed using VEGF-mediated receptor binding, signaling, migration and proliferation. In addition, the ability of TIMP-3 peptides to inhibit CNV in a mouse model was evaluated. We demonstrate that the anti-angiogenic property resides in the COOH-terminal domain of TIMP-3 protein which can block the binding of VEGF specifically to its receptor VEGFR-2, but not to VEGFR-1 similar to the full-length wild-type protein. Synthetic peptides corresponding to putative loop 6 and tail region of TIMP-3 have anti-angiogenic properties as determined by inhibition of VEGF binding to VEGFR-2, VEGF-induced phosphorylation of VEGFR-2 and downstream signaling pathways as well as endothelial cell proliferation and migration in response to VEGF. In addition, we show that intravitreal administration of TIMP-3 peptide could inhibit the size of laser-induced choroidal neovascularization lesions in mice. Thus, we have identified TIMP-3 peptides to be efficient inhibitors of angiogenesis and have a potential to be used therapeutically in diseases with increased neovascularization.