Unregulated hunting and genetic recovery from a severe population decline: the cautionary case of Bulgarian wolves

European wolf (Canis lupus) populations have suffered extensive decline and range contraction due to anthropogenic culling. In Bulgaria, although wolves are still recovering from a severe demographic bottleneck in the 1970s, hunting is allowed with few constraints. A recent increase in hunting pressure has raised concerns regarding long-term viability. We thus carried out a comprehensive conservation genetic analysis using microsatellite and mtDNA markers. Our results showed high heterozygosity levels (0.654, SE 0.031) and weak genetic bottleneck signals, suggesting good recovery since the 1970s decline. However, we found high levels of inbreeding (F _IS  = 0.113, SE 0.019) and a N _e/N ratio lower than expected for an undisturbed wolf population (0.11, 95 % CI 0.08–0.29). We also found evidence for hybridisation and introgression from feral dogs (C. familiaris) in 10 out of 92 wolves (9.8 %). Our results also suggest admixture between wolves and local populations of golden jackals (C. aureus), but less extensive as compared with the admixture with dogs. We detected local population structure that may be explained by fragmentation patterns during the 1970s decline and differences in local ecological characteristics, with more extensive sampling needed to assess further population substructure. We conclude that high levels of inbreeding and hybridisation with other canid species, which likely result from unregulated hunting, may compromise long-term viability of this population despite its current high genetic diversity. The existence of population subdivision warrants an assessment of whether separate management units are needed for different subpopulations. Our study highlights conservation threats for populations with growing numbers but subject to unregulated hunting.     

Influence of exogenous abscisic acid on alterations in protein expression in the proteome of Triticosecale seedlings

The aim of this study was to investigate molecular basis of germination inhibition of Triticosecale under the influence of exogenous ABA. Seedlings were isolated from seeds after 48 h of germination in water (control sample) and 100 μM ABA solution. It was observed that the applied concentration of phytohormone caused a significant inhibition of germination and a suppressed contribution of polysomes in the total ribosomal fraction. To identify proteins whose expression was affected by ABA, a proteomic approach employing 2D electrophoresis was used. Proteome maps for both control and ABA-treated samples displayed over 2,200 Coomassie Brilliant Blue-stained spots each. Protein spots showing either increase or decrease in spot intensity under the ABA influence were identified by mass spectrometry. Many of the identified proteins whose expression was stimulated by ABA proved to be stress-related, involved in nucleotide metabolism or playing a regulatory and signal transduction role, whereas proteins whose expression decreased are known to be involved in numerous metabolic pathways (including energy, carbohydrate or nucleotide metabolism) as well as related to genetic information processing and protein synthesis. Our results show that the germination inhibition caused by ABA is a result of multigene, diversified actions leading together to triticale growth suppression.     

Is CYP1B1 involved in the metabolism of dioxins in the pig?

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most difficult to biodegradate and the most toxic dioxin congener. Previously, we demonstrated in silico the ability of pig CYP1A1 to hydroxylate 2,7-dichlorodibenzo-p-dioxin (DiCDD), but not TCDD. To increase our knowledge concerning the low effectiveness of TCDD biodegradability, we analyzed in silico the binding selectivity and affinity between pig CYP1B1 and the two dioxins by means of molecular modeling. We also compared the effects of TCDD and DiCDD on CYP1B1 gene expression (qRT-PCR) and catalytic (EROD) activity in porcine granulosa cells. It was found that DiCDD and TCDD were stabilized within the pig CYP1B1 active site by hydrophobic interactions. The analysis of substrate channel availability revealed that both dioxins opened the exit channel S, allowing metabolites to leave the enzyme active site. Moreover, DiCDD and TCDD increased the CYP1B1 gene expression and catalytic activity in porcine granulosa cells. On the other hand, TCDD demonstrated higher than DiCDD calculated affinity to pig CYP1B1, hindering TCDD exit from the active site. The great distance between CYP1B1's heme and TCDD also might contribute to the lower hydroxylation effectiveness of TCDD compared to that of DiCDD. Moreover, the narrow active site of pig CYP1B1 may immobilize TCDD molecule, inhibiting its hydroxylation. The results of the access channel analysis and the distance from pig CYP1B1's heme to TCDD suggest that the metabolizing potential of pig CYP1B1 is higher than that of pig CYP1A1. However, this potential is probably not sufficiently high to considerably improve the slow TCDD biodegradation.     

RVX-208, an Inducer of ApoA-I in Humans,Is a BET Bromodomain Antagonist

Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.     

Inhibition of Shiga toxin-converting bacteriophage development by novel antioxidant compounds

Oxidative stress may be the major cause of induction of Shiga toxin-converting (Stx) prophages from chromosomes of Shiga toxin-producing Escherichia coli (STEC) in human intestine. Thus, we aimed to test a series of novel antioxidant compounds for their activities against prophage induction, thus, preventing pathogenicity of STEC. Forty-six compounds (derivatives of carbazole, indazole, triazole, quinolone, ninhydrine, and indenoindole) were tested. Fifteen of them gave promising results and were further characterized. Eleven compounds had acceptable profiles in cytotoxicity tests with human HEK-293 and HDFa cell lines. Three of them (selected for molecular studies) prevent the prophage induction at the level of expression of specific phage genes. In bacterial cells treated with hydrogen peroxide, expression of genes involved in the oxidative stress response was significantly less efficient in the presence of the tested compounds. Therefore, they apparently reduce the oxidative stress, which prevents induction of Stx prophage in E. coli.     

Codon bias and the folding dynamics of the cystic fibrosis transmembrane conductance regulator

Synonymous or silent mutations are often overlooked in genetic analyses for disease-causing mutations unless they are directly associated with potential splicing defects. More recent studies, however, indicate that some synonymous single polynucleotide polymorphisms (sSNPs) are associated with changes in protein expression, and in some cases, protein folding and function. The impact of codon usage and mRNA structural changes on protein translation rates and how they can affect protein structure and function is just beginning to be appreciated. Examples are given here that demonstrate how synonymous mutations alter the translational kinetics and protein folding and/or function. The mechanism for how this occurs is based on a model in which codon usage modulates the translational rate by introducing pauses caused by nonoptimal or rare codons or by introducing changes in the mRNA structure, and this in turn influences co-translational folding. Two examples of this include the multidrug resistance protein (p-glycoprotein) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is also used here as a model to illustrate how synonymous mutations can be examined using in silico predictive methods to identify which sSNPs have the potential to change protein structure. The methodology described here can be used to help identify “non-silent” synonymous mutations in other genes.     

Metallothioneins 1 and 2, but not 3, are regulated by nutritional status in rat white adipose tissue

Background

Cumulating evidence underlines the role of adipose tissue metallothionein (MT) in the development of obesity and type 2 diabetes. Fasting/refeeding was shown to affect MT gene expression in the rodent liver. The influence of nutritional status on MT gene expression in white adipose tissue (WAT) is inconclusive. The aim of this study was to verify if fasting and fasting/refeeding may influence expression of MT genes in WAT of rats.

Results

Fasting resulted in a significant increase in MT1 and MT2 gene expressions in retroperitoneal, epididymal, and inguinal WAT of rats, and this effect was reversed by refeeding. Altered expressions of MT1 and MT2 genes in all main fat depots were reflected by changes in serum MT1 and MT2 levels. MT1 and MT2 messenger RNA (mRNA) levels in WAT correlated inversely with serum insulin concentration. Changes in MT1 and MT2 mRNA levels were apparently not related to total zinc concentrations and MTF1 and Zn transporter mRNA levels in WAT. Fasting or fasting/refeeding exerted no effect on the expression of MT3 gene in WAT. Addition of insulin to isolated adipocytes resulted in a significant decrease in MT1 and MT2 gene expressions. In contrast, forskolin or dibutyryl-cAMP (dB-cAMP) enhanced the expressions of MT1 and MT2 genes in isolated adipocytes. Insulin partially reversed the effect of dB-cAMP on MT1 and MT2 gene expressions.

Conclusions

This study showed that the expressions of MT1 and MT2 genes in WAT are regulated by nutritional status, and the regulation may be independent of total zinc concentration.

     

HPLC columns partition by chemometric methods based on peptides retention

In recent years, multivariate techniques have been utilized to evaluate reversed-phase high-performance liquid chromatographic data. In the present study, 11 high-performance liquid chromatography (HPLC) columns were divided into several groups according to the retention factors of 12 peptides. Principal component analysis (PCA) and cluster analysis (CA) were used in column and peptides' comparison and grouping. CA results indicated that all stationary phases may be generally grouped into several clusters, due to stationary phase structure and properties. On the other hand, interesting results were obtained with the use of PC. There is almost linear relationship between classified HPLC columns in the space of new PCs, which is connected with meaning of the PC's reflected in their loading values. The first component describes non-polar properties of peptides, whereas the second component is loaded with polar peptides having much lower logP values. PCA and CA were also used in peptides comparison however, complete explanation of peptides grouping still remains unclear.