A noninvasive, direct real-time PCR method for sex determination in multiple avian species

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis.     

Behavioral transitions and weight change patterns within the PREMIER trial

Little is known about the transition in behaviors from short-term weight loss to maintenance of weight loss. We wanted to determine how short-term and long-term weight loss and patterns of weight change were associated with intervention behavioral targets. This analysis includes overweight/obese participants in active treatment (n = 507) from the previously published PREMIER trial, an 18-month, multicomponent lifestyle intervention for blood pressure reduction, including 33 intervention sessions and recommendations to self-monitor food intake and physical activity daily. Associations between behaviors (attendance, recorded days/week of physical activity, food records/week) and weight loss of ≥5% at 6 and 18 months were examined using logistic regression. We characterized the sample using 5 weight change categories (weight gained, weight stable, weight loss then relapse, late weight loss, and weight loss then maintenance) and analyzed adherence to the behaviors for each category, comparing means with ANOVA. Participants lost an average of 5.3 ± 5.6 kg at 6 months and 4.0 ± 6.7 kg (4.96% of body weight) by 18 months. Higher levels of attendance, food record completion, and recorded days/week of physical activity were associated with increasing odds of achieving 5% weight loss. All weight change groups had declines in the behaviors over time; however, compared to the other four groups, the weight loss/maintenance group (n = 154) had statistically less significant decline in number of food records/week (48%), recorded days/week of physical activity (41.7%), and intervention sessions attended (12.8%) through 18 months. Behaviors associated with short-term weight loss continue to be associated with long-term weight loss, albeit at lower frequencies. Minimizing the decline in these behaviors may be important in achieving long-term weight loss.     

Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals

Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4-18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance.     

Effects of engineered nanoparticles on the assembly of exopolymeric substances from phytoplankton

The unique properties of engineered nanoparticles (ENs) that make their industrial applications so attractive simultaneously raise questions regarding their environmental safety. ENs exhibit behaviors different from bulk materials with identical chemical compositions. Though the nanotoxicity of ENs has been studied intensively, their unintended environmental impacts remain largely unknown. Herein we report experimental results of EN interactions with exopolymeric substances (EPS) from three marine phytoplankton species: Amphora sp., Ankistrodesmus angustus and Phaeodactylum tricornutum. EPS are polysaccharide-rich anionic colloid polymers released by various microorganisms that can assemble into microgels, possibly by means of hydrophobic and ionic mechanisms. Polystyrene nanoparticles (23 nm) were used in our study as model ENs. The effects of ENs on EPS assembly were monitored with dynamic laser scattering (DLS). We found that ENs can induce significant acceleration in Amphora sp. EPS assembly; after 72 hours EN-EPS aggregation reached equilibrium, forming microscopic gels of ～4-6 μm in size. In contrast, ENs only cause moderate assembly kinetic acceleration for A. angustus and P. tricornutum EPS samples. Our results indicate that the effects of ENs on EPS assembly kinetics mainly depend on the hydrophobic interactions of ENs with EPS polymers. The cycling mechanism of EPS is complex. Nonetheless, the change of EPS assembly kinetics induced by ENs can be considered as one potential disturbance to the marine carbon cycle.     

Nutritional geometry: gorillas prioritize non-protein energy while consuming surplus protein

It is widely assumed that terrestrial food webs are built on a nitrogen-limited base and consequently herbivores must compensate through selection of high-protein foods and efficient nitrogen retention. Like many folivorous primates, gorillas' diet selection supports this assumption, as they apparently prefer protein-rich foods. Our study of mountain gorillas (Gorilla beringei) in Uganda revealed that, in some periods, carbohydrate-rich fruits displace a large portion of protein-rich leaves in their diet. We show that non-protein energy (NPE) intake was invariant throughout the year, whereas protein intake was substantially higher when leaves were the major portion of the diet. This pattern of macronutrient intake suggests that gorillas prioritize NPE and, to achieve this when leaves are the major dietary item, they over-eat protein. The concentrations of protein consumed in relation to energy when leaves were the major portion of the diet were close to the maximum recommended for humans and similar to high-protein human weight-loss diets. By contrast, the concentrations of protein in relation to energy when gorillas ate fruit-dominated diets were similar to those recommended for humans. Our results question the generality of nitrogen limitation in terrestrial herbivores and provide a fascinating contrast with human macronutrient intake.     

Leeches run cold, then hot

Food processing is costly, potentially limiting the energy and time devoted to other essential functions such as locomotion or reproduction. In ectotherms, post-prandial thermophily, the selection of a warm environmental temperature after feeding, may be advantageous in minimizing the duration of this elevated cost. Although present in many vertebrate taxa, this behaviour had not previously been observed in invertebrates. Sanguivorous leeches ingest large blood meals that are costly to process and limit mobility until excess fluid can actively be expelled to reduce body volume. When presented with a temperature gradient from 10°C to 30°C, leeches select a temperature that is significantly warmer (24.3 ± 0.9°C, n = 6) than their acclimation temperature (T(a), 21°C). Unfed leeches preferred temperatures that were significantly cooler than ambient (12.8 ± 0.9°C, n = 6). This behavioural strategy is consistent with minimizing the time course of elevated post-feeding energy costs and reducing energy expenditure during fasting. Our observations raise the possibility that thermoregulatory behaviour of this type is an unrecognized feature of other invertebrate taxa.     

A comprehensive genome‐scale reconstruction of Escherichia coli metabolism—2011

The initial genome‐scale reconstruction of the metabolic network of Escherichia coli K‐12 MG1655 was assembled in 2000. It has been updated and periodically released since then based on new and curated genomic and biochemical knowledge. An update has now been built, named iJO1366, which accounts for 1366 genes, 2251 metabolic reactions, and 1136 unique metabolites. iJO1366 was (1) updated in part using a new experimental screen of 1075 gene knockout strains, illuminating cases where alternative pathways and isozymes are yet to be discovered, (2) compared with its predecessor and to experimental data sets to confirm that it continues to make accurate phenotypic predictions of growth on different substrates and for gene knockout strains, and (3) mapped to the genomes of all available sequenced E. coli strains, including pathogens, leading to the identification of hundreds of unannotated genes in these organisms. Like its predecessors, the iJO1366 reconstruction is expected to be widely deployed for studying the systems biology of E. coli and for metabolic engineering applications.     

Social influences are associated with BMI and weight loss intentions in young adults

Christakis and colleagues have shown that health behaviors cluster in social networks and suggest social norms may account for the clustering. This study examined: (i) whether obesity clusters among young adults and whether social norms do in fact account for the clustering, and (ii) among overweight/obese (OW/OB) young adults, whether number of social contacts trying to lose weight is associated with weight loss intentions and whether social norms for weight loss account for this effect. Normal weight (NW) and OW/OB young adults (N = 288; 66% female; 75% white) completed measures assessing number of OW social contacts and social norms for obesity. OW/OB young adults also indicated number of OW social contacts currently trying to lose weight, social norms for weight loss, and weight loss intentions. Compared to NW, OW/OB young adults were more likely to have OW romantic partners and best friends and had more OW casual friends and family members (Ps < 0.05), but social norms for obesity did not differ between groups, and social norms did not mediate the relationship between OW social contacts and participants' weight status. However, among OW/OB young adults, having more social contacts trying to lose weight was associated with greater intention to lose weight (r = 0.20, P = 0.02) and social norms for weight loss fully mediated this effect (P < 0.01). This study is the first to show that social contacts and normative beliefs influence weight status and intentions for weight control in young adults. Findings underscore the importance of targeting social influence in the treatment and prevention of obesity in this high-risk age group.     

Adeno-associated virus-mediated rescue of the cognitive defects in a mouse model for Angelman syndrome

Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII) regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr(286) and Thr(305/306), resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV) vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.     

Akt1 is essential for postnatal mammary gland development, function, and the expression of Btn1a1

Akt1, a serine-threonine protein kinase member of the PKB/Akt gene family, plays critical roles in the regulation of multiple cellular processes, and has previously been implicated in lactation and breast cancer development. In this study, we utilized Akt1+/+ and Akt1−/− C57/Bl6 female mice to assess the role that Akt1 plays in normal mammary gland postnatal development and function. We examined postnatal morphology at multiple time points, and analyzed gene and protein expression changes that persist into adulthood. Akt1 deficiency resulted in several mammary gland developmental defects, including ductal outgrowth and defective terminal end bud formation. Adult Akt1−/− mammary gland composition remained altered, exhibiting fewer alveolar buds coupled with increased epithelial cell apoptosis. Microarray analysis revealed that Akt1 deficiency altered expression of genes involved in numerous biological processes in the mammary gland, including organismal development, cell death, and tissue morphology. Of particular importance, a significant decrease in expression of Btn1a1, a gene involved in milk lipid secretion, was observed in Akt1−/− mammary glands. Additionally, pseudopregnant Akt1−/− females failed to induce Btn1a1 expression in response to hormonal stimulation compared to their wild-type counterparts. Retroviral-mediated shRNA knockdown of Akt1 and Btn1a1 in MCF-7 human breast epithelial further illustrated the importance of Akt1 in mammary epithelial cell proliferation, as well as in the regulation of Btn1a1 and subsequent expression of ß-casein, a gene that encodes for milk protein. Overall these findings provide mechanistic insight into the role of Akt1 in mammary morphogenesis and function.