Estimating amplification efficiency improves multiplex real-time PCR quantification of Bacillus licheniformis and Bacillus subtilis spores in animal feed

A multiplex real-time PCR assay was developed for absolute quantification in animal feed of Bacillus subtilis CH201 and Bacillus licheniformis CH200 spores, which constitute the viable component of the microbial growth promoter, BioPlus 2B. Spores were lysed using a bead-beating protocol. DNA was extracted and purified from the lysates with the Qiagen DNeasy Plant Kit. Two standard curves for absolute quantification were made and tested. Standard curve-1 was made from feed samples spiked with BioPlus 2B, while standard curve-2 was made from serially diluted DNA extracted from BioPlus 2B powder. Feed samples supplemented with BioPlus 2B were quantified using both standard curves. The detection limit of the assay was 10(4) CFU g(-1) of feed. The amplification efficiency (Eff) of each PCR was determined using the LinRegPCR software and Eff differences between individual samples and standards were corrected for. When compared to plate counts, standard curve-1 slightly under-estimated the number of spores (mean=-2.47% of plate counts). A spore density-dependent Eff was found, and Eff for standard curve-1 could not be determined. Standard curve-2 over-estimated spore numbers when not corrected for individual Eff (mean=+5.46% of plate counts). Standard curve-2 Eff was independent (Eff(mean)=1.96) of spore density. The assay quantified the numbers of spores in feed samples very similar to plate counts (mean=+0.47% of plate counts), when standard curve-2 was used and individual Eff was accounted for.     

Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states

Skeletal muscle has the ability to achieve rapid repair in response to injury or disease. Many individuals with Marfan syndrome (MFS), caused by a deficiency of extracellular fibrillin-1, exhibit myopathy and often are unable to increase muscle mass despite physical exercise. Evidence suggests that selected manifestations of MFS reflect excessive signaling by transforming growth factor (TGF)-beta (refs. 2,3). TGF-beta is a known inhibitor of terminal differentiation of cultured myoblasts; however, the functional contribution of TGF-beta signaling to disease pathogenesis in various inherited myopathic states in vivo remains unknown. Here we show that increased TGF-beta activity leads to failed muscle regeneration in fibrillin-1-deficient mice. Systemic antagonism of TGF-beta through administration of TGF-beta-neutralizing antibody or the angiotensin II type 1 receptor blocker losartan normalizes muscle architecture, repair and function in vivo. Moreover, we show TGF-beta-induced failure of muscle regeneration and a similar therapeutic response in a dystrophin-deficient mouse model of Duchenne muscular dystrophy.     

Antibacterial functionalization of an experimental self-etching primer by inorganic agents: microbiological and biocompatibility evaluations

Antibacterial activities have been demonstrated on oral bacteria with inorganic antibacterial agents (ABAs) after their incorporations into an experimental self-etching primer (ESP) before curing. This study was to assess their biocompatibility and antibacterial activity after curing. Six ABAs were incorporated respectively into ESP for treating specimens. After curing, their bactericidal activities on Streptococcus mutans and influences to the early bacterial colonization were assessed by direct contact and viable count. Systemic toxicity in rats after short-term oral exposure and direct contact cytotoxicity with NIH3T3 fibroblasts were tested. Incorporation of ZnOw AT-83, Longbei antibiotic, Antim-AMS2 or IONPURE-H significantly enhanced the antibacterial effect of ESP after curing, even after 1 month aging. Specimens treated by ESP with ZnOw AT-83, Longbei antibiotic or Antim-AMS2 showed slightly less bacterial adhesion than control. Animal experiments revealed neither toxic signs nor significant differences in body weight gain between control and other groups. Cell vitality or proliferation rates were ranged from 76% to 100% with respect to controls. Basic magnesium hypochlorite, ZnOw AT-83 and ZnOw AT-88 were less toxic. Toxicity only observed in areas beneath the specimens and/or in the direct vicinity of the specimen edge. From microbiological and biocompatibility aspects, the tested ABAs can be effectively incorporated in ESP to provide antibacterial activity against S. mutans. ZnOw AT-83 was the most promising one.     

Operationalising emission and toxicity modelling of pesticides in LCA: the OLCA-Pest project contribution

The International Journal of Life Cycle Assessment - Current field emission modelling and toxicity characterisation of pesticides suffer from several shortcomings like mismatches between LCI...     

Impact of QTL properties on the accuracy of multi-breed genomic prediction

Background

Although simulation studies show that combining multiple breeds in one reference population increases accuracy of genomic prediction, this is not always confirmed in empirical studies. This discrepancy might be due to the assumptions on quantitative trait loci (QTL) properties applied in simulation studies, including number of QTL, spectrum of QTL allele frequencies across breeds, and distribution of allele substitution effects. We investigated the effects of QTL properties and of including a random across- and within-breed animal effect in a genomic best linear unbiased prediction (GBLUP) model on accuracy of multi-breed genomic prediction using genotypes of Holstein-Friesian and Jersey cows.

Methods

Genotypes of three classes of variants obtained from whole-genome sequence data, with moderately low, very low or extremely low average minor allele frequencies (MAF), were imputed in 3000 Holstein-Friesian and 3000 Jersey cows that had real high-density genotypes. Phenotypes of traits controlled by QTL with different properties were simulated by sampling 100 or 1000 QTL from one class of variants and their allele substitution effects either randomly from a gamma distribution, or computed such that each QTL explained the same variance, i.e. rare alleles had a large effect. Genomic breeding values for 1000 selection candidates per breed were estimated using GBLUP modelsincluding a random across- and a within-breed animal effect.

Results

For all three classes of QTL allele frequency spectra, accuracies of genomic prediction were not affected by the addition of 2000 individuals of the other breed to a reference population of the same breed as the selection candidates. Accuracies of both single- and multi-breed genomic prediction decreased as MAF of QTL decreased, especially when rare alleles had a large effect. Accuracies of genomic prediction were similar for the models with and without a random within-breed animal effect, probably because of insufficient power to separate across- and within-breed animal effects.

Conclusions

Accuracy of both single- and multi-breed genomic prediction depends on the properties of the QTL that underlie the trait. As QTL MAF decreased, accuracy decreased, especially when rare alleles had a large effect. This demonstrates that QTL properties are key parameters that determine the accuracy of genomic prediction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0124-6) contains supplementary material, which is available to authorized users.     

Identification of chemically diverse, novel inhibitors of 17β-hydroxysteroid dehydrogenase type 3 and 5 by pharmacophore-based virtual screening

17β-Hydroxysteroid dehydrogenase type 3 and 5 (17β-HSD3 and 17β-HSD5) catalyze testosterone biosynthesis and thereby constitute therapeutic targets for androgen-related diseases or endocrine-disrupting chemicals. As a fast and efficient tool to identify potential ligands for 17βHSD3/5, ligand- and structure-based pharmacophore models for both enzymes were developed. The models were evaluated first by in silico screening of commercial compound databases and further experimentally validated by enzymatic efficacy tests of selected virtual hits. Among the 35 tested compounds, 11 novel inhibitors with distinct chemical scaffolds, e.g. sulfonamides and triazoles, and with different selectivity properties were discovered. Thereby, we provide several potential starting points for further 17β-HSD3 and 17β-HSD5 inhibitor development. Article from the Special issue on Targeted Inhibitors.