Association between regulator of G protein signaling 9-2 and body weight

Regulator of G protein signaling 9-2 (RGS9-2) is a protein that is highly enriched in the striatum, a brain region that mediates motivation, movement and reward responses. We identified a naturally occurring 5 nucleotide deletion polymorphism in the human RGS9 gene and found that the mean body mass index (BMI) of individuals with the deletion was significantly higher than those without. A splicing reporter minigene assay demonstrated that the deletion had the potential to significantly decrease the levels of correctly spliced RGS9 gene product. We measured the weights of rats after virally transduced overexpression of RGS9-2 or the structurally related RGS proteins, RGS7, or RGS11, in the nucleus accumbens (NAc) and observed a reduction in body weight after overexpression of RGS9-2 but not RGS7 or 11. Conversely, we found that the RGS9 knockout mice were heavier than their wild-type littermates and had significantly higher percentages of abdominal fat. The constituent adipocytes were found to have a mean cross-sectional area that was more than double that of corresponding cells from wild-type mice. However, food intake and locomotion were not significantly different between the two strains. These studies with humans, rats and mice implicate RGS9-2 as a factor in regulating body weight.     

Adjustment of juvenile tuatara to a cooler,southern climate: operative temperatures,emergence behaviour and growth rate

Translocations for conservation often involve species limited to relict distributions. However, uncertainty can exist regarding the ability of source individuals to acclimatise following a shift to a distant location. We investigated the ability of captive-reared juvenile tuatara (Sphenodon punctatus) of Cook Strait stock (41°S) to adjust to outdoor, predator-protected pens within Orokonui Ecosanctuary (45 °S). We examined potential basking and within burrow temperatures, the influence of temperature on emergence, and growth rates in comparison with other locations. Tuatara at Orokonui reached their preferred temperature when basking over summer, and burrows provided protection from freezing over winter. Emergence was temperature-dependent and essentially ceased during winter. Growth rates of Orokonui-held juveniles were within the range for four other captive-rearing facilities and faster than for wild juveniles from a Cook Strait population. As all Orokonui-held juveniles have survived and grown we conclude that the climate at this southern location is suitable to consider a free-release.     

Enteral calcium infusion used successfully as treatment for a patient with hereditary vitamin D resistant rickets (HVDRR) without alopecia: A novel mutation

Background

We report a novel mutation in a case of hereditary vitamin D resistant rickets (HVDRR) without alopecia and successful management of this condition with the intravenous formulation of calcium chloride delivered via gastric tube.

Clinical Case

A 22 month old male (length − 3.4 SDS; weight − 2.1 SDS) presented with failure to thrive, short stature, severe hypocalcemia and gross motor delay. He did not have alopecia. Initial blood tests and history were thought possibly to suggest vitamin D deficiency rickets: calcium 5.1 mg/dL, (8.8–10.8); phosphorus 4.1 mg/dL, (4.5–5.5); alkaline phosphatase 1481 U/L (80–220); intact PTH 537.1 pg/mL (10–71). Subsequently, vitamin D studies returned that were consistent with HVDRR: 25-hydroxyvitamin D 34 ng/mL (20–100); 1,25-dihydroxyvitamin D 507 pg/mL. This diagnosis was confirmed by DNA sequencing. His subsequent clinical course was complicated by the fact that IV calcium was not a viable option for this patient, and his calcium levels could not be well controlled on oral calcium citrate or calcium glubionate therapy. Eventually, we were able to maintain calcium levels above 8 mg/dL using the intravenous preparation of calcium chloride administered via gastric tube.

Genetic Studies

A unique homozygous T to C base substitution was found in exon 6 in the vitamin D receptor (VDR) gene. This mutation causes leucine to be converted to proline at position 227 in helix 3 in the VDR ligand binding domain (LBD). The mutation rendered the VDR non-functional, leading to HVDRR, with absence of alopecia.

Conclusion

HVDRR should be considered in a patient with profound hypocalcemia which is refractory to conventional therapy of vitamin D deficiency rickets even without evidence of alopecia. We report the first case of HVDRR with a novel mutation in the LBD that was successfully treated with enteral treatment using a calcium chloride infusion.     

Impact of variance components on reliability of absolute quantification using digital PCR

Background

Digital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation.

Results

We reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably.

Conclusions

Like any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-283) contains supplementary material, which is available to authorized users.     

Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse human glioblastomas in the mouse brain in vivo

Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused (13)C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.     

WW-domain-containing oxidoreductase is associated with low plasma HDL-C levels

Low serum HDL-cholesterol (HDL-C) is a major risk factor for coronary artery disease. We performed targeted genotyping of a 12.4 Mb linked region on 16q to test for association with low HDL-C by using a regional-tag SNP strategy. We identified one SNP, rs2548861, in the WW-domain-containing oxidoreductase (WWOX) gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent (p = 6.9 × 10⁻⁷). We extended our investigation to the population level by using two independent unascertained population-based Finnish cohorts, the cross-sectional METSIM cohort of 4,463 males and the prospective Young Finns cohort of 2,265 subjects. The combined analysis provided p = 4 × 10⁻⁴ to 2 × 10⁻⁵. Importantly, in the prospective cohort, we observed a significant longitudinal association of rs2548861 with HDL-C levels obtained at four different time points over 21 years (p = 0.003), and the T risk allele explained 1.5% of the variance in HDL-C levels. The rs2548861 resides in a highly conserved region in intron 8 of WWOX. Results from our in vitro reporter assay and electrophoretic mobility-shift assay demonstrate that this region functions as a cis-regulatory element whose associated rs2548861 SNP has a specific allelic effect and that the region forms an allele-specific DNA-nuclear-factor complex. In conclusion, analyses of 9,798 subjects show significant association between HDL-C and a WWOX variant with an allele-specific cis-regulatory function.     

Diversity of seeds captured by interception exceeds diversity of seeds deposited in traps

Seed dispersal, a key process in terrestrial landscapes, is increasingly important in the face of habitat fragmentation and global climate change. Seed dispersal is also notoriously difficult to characterize, especially in species rich and spatially complex tropical forests. We contrasted assemblages of biotically dispersed seeds collected from four sites using two methods: deposition into seed traps and interception by the capture of frugivorous birds. We also compared seed deposition and interception with local fruit production. Species accumulation curves for seeds deposited in seed traps began to level off sooner than curves for seeds collected from birds captured in mist nets, and extrapolation showed significantly greater estimated species richness for seeds collected from birds than for those deposited in traps. Assemblages from birds and from traps at each site were quite different, with an abundance‐based similarity index of 0.64; this dissimilarity increases if bat‐dispersed seeds are included in the analysis. Common bird‐dispersed species were retrieved from both mist‐netted birds and from seed traps, but numerous locally fruiting understory species were recovered only from birds. We conclude that the sampling of seeds carried by birds provides a valuable complement to other methods of studying seed dispersal in species‐rich tropical forests by revealing relationships between specific dispersers and their seed plants and by creating a more complete account of species diversity of seeds being transported at a given site.     

“How” and “Why” prompts in forensic investigative interviews with preschool children

Although young children may frequently be asked “How” and “Why” questions, it is unclear whether they have the ability to respond well enough to justify the use of these words during investigative interviews. The range of possible uses and interpretations of the words “How” and “Why” makes it critical to examine their use when communicatively immature children are interviewed. In this study, police interviews of 3- to 5-year-old suspected victims of sexual abuse (n = 49) were examined. The use of How/Why prompts by interviewers and children’s responses to interviewers’ How/Why prompts were coded. How/Why prompts represented 22% of all interviewer prompts. Of all details provided by children, however, 8.5% were in response to How/Why prompts. In addition, children provided the information sought in response to only 20% of the interviewers’ How/Why prompts, whereas uninformative responses were relatively common. Children responded to more How/Why prompts with the information sought by interviewers as they grew older. The findings suggest that How/Why prompts may not be particularly effective when interviewing preschool children.