Human familial and sporadic breast cancer: analysis of the coding regions of the 17β-hydroxysteroid dehydrogenase 2 gene (EDH17B2) using a single-strand conformation polymorphism assay

17-Hydroxysteroid dehydrogenase (17HSD) is one of the key enzymes in estrogen metabolism, catalyzing the reversible reaction between estradiol and the less active estrogen, estrone. The gene encoding this enzyme, EDH17B2, has been mapped to chromosome 17, region q12–q21, in the vicinity of BRCA1, an as yet unidentified gene that appears to be involved in familial breast cancer and in familial ovarian cancer. The possibility that EDH17B2 gene is the same as BRCA1 was tested by screening for mutations in the coding regions of EDH17B2, using a polymerase chain reaction/single-strand conformation polymorphism method. An AG transition creating a new BstUI site at exon 6 was the only frequent sequence alteration found in the coding region of the gene. This mutation also led to an amino acid substitution of serine to glycine at position 312 (312^S312^G) in the 17HSD protein. Since the nucleotide change was detected both in specimens from patients with familial or sporadic cancer and in control samples, and at similar rates, this mutation appears to be of a polymorphic nature. In addition, a rare polymorphism located at intron 5 was detected. This CT substitution creates a BbvI site and is not thought to have any effect on 17HSD activity. The results indicate that there are no major alterations in the coding areas of EDH17B2 and thus studies testing the hypothesis that EDH17B2 may be the same as BRCA1 should be extended to the promoter and regulatory elements of EDH17B2.     

Growth performance and feed conversion of Ruspolia differens on plant-based by-product diets

The use of food industry by-products in insect feeds has gained increasing attention recently. However, the understanding of how well the economically valuable edible insect Ruspolia differens (Serville) (Orthoptera: Tettigoniidae) can grow and develop with plant-based by-product feeds is currently lacking. It is important to determine the nutritional requirements, especially protein demand, of this species before developing artificial feeds for mass-rearing. We reared R. differens with four control diets and 12 plant-based by-product diets in which the major protein source came from food industry by-products, including potato-protein, barley mash, barley feed, turnip rape, a mix of broad bean and pea, and a mix of potato, carrot, and apple. We asked whether the performance (development time, survival, and weight), feed conversion, and fatty acid composition and content differed among diet treatments. Furthermore, the 12 experimental by-product diets were designed to reach six protein levels. We found that R. differens can be reared with various by-product diets, but development time, survival, and weight differed among diets. Barley feed, barley mash, and potato protein diets seem to be good options for rearing, and potato glycoalkaloids do not affect the performance of R. differens. Individuals fed on the various by-product diets also differed in their fatty acid composition and content. Increasing protein levels in diet up to 17% enhanced growth, development time, and survival, but no further enhancements were seen when fed diets with protein levels higher than this. The high protein levels decreased feed conversion rate. Our results can be valuable for designing feeds for insect mass-rearing technology. The use of food industry by-products in the diets for R. differens could increase the re-use of local resources and enhance circular economy.     

Female-specific resource limitation does not make the opportunity for selection more female biased

Competition for limiting resources and stress can magnify variance in fitness and therefore selection. But even in a common environment, the strength of selection can differ across the sexes, as their fitness is often limited by different factors. Indeed, most taxa show stronger selection in males, a bias often ascribed to intense competition for access to mating partners. This sex bias could reverberate on many aspects of evolution, from speed of adaptation to genome evolution. It is unclear, however, whether stronger opportunity for selection in males is a pattern robust to sex-specific stress or resource limitation. We test this in the model species Callosobruchus maculatus by comparing female and male opportunity for selection (i) with and without limitation of quality oviposition sites, and (ii) under delayed age at oviposition. Decreasing the abundance of the resource key to females or increasing their reproductive age was challenging, as shown by a reduction in mean fitness, but opportunity for selection remained stronger in males across all treatments, and even more so when oviposition sites were limiting. This suggests that males remain the more variable sex independent of context, and that the opportunity for selection through males is indirectly affected by female-specific resource limitation.     

All-Cause and Cause-Specific Mortality among Users of Basal Insulins NPH,Detemir, and Glargine

Background

Insulin therapy in type 2 diabetes may increase mortality and cancer incidence, but the impact of different types of basal insulins on these endpoints is unclear. Compared to the traditional NPH insulin, the newer, longer-acting insulin analogues detemir and glargine have shown benefits in randomized controlled trials. Whether these advantages translate into lower mortality among users in real life is unknown.

Objective

To estimate the differences in all-cause and cause-specific mortality rates between new users of basal insulins in a population-based study in Finland.

Methods

23 751 individuals aged ≥40 with type 2 diabetes, who initiated basal insulin therapy in 2006–2009 were identified from national registers, with comprehensive data for mortality, causes of death, and background variables. Propensity score matching was performed on characteristics. Follow-up time was up to 4 years (median 1.7 years).

Results

2078 deaths incurred. With NPH as reference, the adjusted HRs for all-cause mortality were 0.39 (95% CI, 0.30–0.50) for detemir, and 0.55 (95% CI, 0.44–0.69) for glargine. As compared to glargine, the HR was 0.71 (95% CI, 0.54–0.93) among detemir users. Compared to NPH, the mortality risk for both cardiovascular causes as well as cancer were also significantly lower for glargine, and especially for detemir in adjusted analysis. Furthermore, the results were robust in various sensitivity analyses.

Conclusion

In real clinical practice, mortality was substantially higher among users of NPH insulin as compared to insulins detemir or glargine. Considering the large number of patients who require insulin therapy, this difference in risk may have major clinical and public health implications. Due to limitations of the observational study design, further investigation using an interventional study design is warranted.     

Factors that Influence the Formation and Stability of Thin,Cryo-EM Specimens

Poor consistency of the ice thickness from one area of a cryo-electron microscope (cryo-EM) specimen grid to another, from one grid to the next, and from one type of specimen to another, motivates a reconsideration of how to best prepare suitably thin specimens. Here we first review the three related topics of wetting, thinning, and stability against dewetting of aqueous films spread over a hydrophilic substrate. We then suggest that the importance of there being a surfactant monolayer at the air-water interface of thin, cryo-EM specimens has been largely underappreciated. In fact, a surfactant layer (of uncontrolled composition and surface pressure) can hardly be avoided during standard cryo-EM specimen preparation. We thus suggest that better control over the composition and properties of the surfactant layer may result in more reliable production of cryo-EM specimens with the desired thickness.     

Quaternary structure built from subunits combining NMR and small-angle x-ray scattering data

A new principle in constructing molecular complexes from the known high-resolution domain structures joining data from NMR and small-angle x-ray scattering (SAXS) measurements is described. Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The residual dipolar coupling data serves to reduce angular degrees of freedom, and the small-angle scattering data serves to confine the translational degrees of freedom. The complex built by this method was found to be consistent with the known crystal structure. The study demonstrates how approximate tertiary structures of modular proteins or quaternary structures composed of subunits can be assembled from high-resolution structures of domains or subunits using mutually complementary NMR and SAXS data.     

Family-Based Cluster Randomized Controlled Trial Enhancing Physical Activity and Motor Competence in 4–7-Year-Old Children

Little is known of how to involve families in physical activity (PA) interventions for children. In this cluster randomized controlled trial, we recruited families with four- to seven-year-old children to participate in a year-long study where parents in the intervention group families (n = 46) received tailored counseling to increase children’s PA. Structured PA was not served. Control group families (n = 45) did not receive any counseling. PA in all children (n = 91; mean age 6.16 ± 1.13 years at the baseline) was measured by accelerometers at the baseline and after three, six, nine and 12 months. Motor competence (MC) (n = 89) was measured at the baseline and after six and 12 months by a KTK (KörperkoordinationsTest für Kinder) and throwing and catching a ball (TCB) protocols. The effect of parental counseling on study outcomes was analyzed by a linear mixed-effects model fit by REML and by a Mann-Whitney U test in the case of the TCB. As season was hypothesized to affect counseling effect, an interaction of season on the study outcomes was examined. The results show significant decrease of MVPA in the intervention group when compared to the control group (p < .05). The TCB showed a nearly significant improvement at six months in the intervention group compared to the controls (p = .051), but not at 12 months. The intervention group had a steadier development of the KTK when the interaction of season was taken into account. In conclusion, more knowledge of family constructs associating with the effectiveness of counseling is needed for understanding how to enhance PA in children by parents. However, a hypothesis may be put forward that family-based counseling during an inactive season rather than an active season may provide a more lasting effect on the development of KTK in children.

Trial Registration

Controlled-Trials.com ISRCTN28668090     

Recombination Enhances HIV-1 Envelope Diversity by Facilitating the Survival of Latent Genomic Fragments in the Plasma Virus Population

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation process including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.     

Ribosome reactivation by replacement of damaged proteins

Ribosomal functions are vital for all organisms. Bacterial ribosomes are stable 2.4 MDa particles composed of three RNAs and over 50 different proteins. Accumulating damage to ribosomal RNA or proteins can disturb ribosome functioning. Organisms could benefit from degrading or possibly repairing inactive or partially active ribosomes. Reactivation of chemically damaged ribosomes by a process of protein replacement was studied in vitro. Ribosomes were inactivated by chemical modification of Cys residues. Incubation of modified ribosomes with total ribosomal proteins led to reactivation of translational activity. Intriguingly, ribosomal proteins extracted by LiCl are equally active in the restoration of ribosome function. Incubation of 70S ribosomes with isotopically labelled r‐proteins followed by separation of ribosomes was used to identify exchangeable proteins. A similar set of proteins was found to be exchanged in vivo under stress conditions in the stationary phase. We propose that repair of damaged ribosomes might be an important mechanism for maintaining protein synthesis activity following chemical damage.