Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use

Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex‐linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex‐reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress‐induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North‐Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female‐to‐male sex‐reversed adults had similar body mass as normal males. We recorded no events of male‐to‐female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human‐induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex‐reversed individuals surviving to adulthood may participate in breeding.     

Matrix metalloproteinases and soluble Fas/FasL system as novel regulators of apoptosis in children and young adults on chronic dialysis

The system of membrane receptor Fas and its ligand FasL compose one of the main pathways triggering apoptosis. However, the role of their soluble forms has not been clarified yet. Although sFasL can be converted from the membrane-bound form by matrix metalloproteinases (MMPs), there are no data on relations between sFas/sFasL, MMPs and their tissue inhibitors (TIMPs) in patients on chronic dialysis—neither children nor adults. The aim of our study was to evaluate serum concentrations of sFas, sFasL, and their potential regulators (MMP-2, MMP-7, MMP-9, TIMP-1, TIMP-2), in children and young adults chronically dialyzed. Twenty-two children on automated peritoneal dialysis (APD), 19 patients on hemodialysis (HD) and 30 controls were examined. Serum concentrations of sFas, sFasL, MMPs and TIMPs were assessed by ELISA. Median values of sFas, sFasL, sFas/sFasL ratio, MMP-2, MMP-7, MMP-9, TIMP-1 and TIMP-2 were significantly elevated in all dialyzed patients vs. controls, the highest values being observed in subjects on HD. A single HD session caused the decrease in values of all parameters to the levels below those seen in children on APD. Regression analysis revealed that MMP-7 and TIMP-1 were the best predictors of sFas and sFasL concentrations. Children and young adults on chronic dialysis are prone to sFas/sFasL system dysfunction, more pronounced in patients on hemodialysis. The correlations between sFas/sFasL and examined enzymes suggest that MMPs and TIMPs take part in the regulation of cell death in the pediatric population on chronic dialysis, triggering both anti- (sFas) and pro-apoptotic (sFasL) mechanisms.     

Shugoshin is a Mad1/Cdc20-like interactor of Mad2

Mammalian centromeric cohesin is protected from phosphorylation-dependent displacement in mitotic prophase by shugoshin-1 (Sgo1), while shugoshin-2 (Sgo2) protects cohesin from separase-dependent cleavage in meiosis I. In higher eukaryotes, progression and faithful execution of both mitosis and meiosis are controlled by the spindle assembly checkpoint, which delays anaphase onset until chromosomes have achieved proper attachment to microtubules. According to the so-called template model, Mad1-Mad2 complexes at unattached kinetochores instruct conformational change of soluble Mad2, thus catalysing Mad2 binding to its target Cdc20. Here, we show that human Sgo2, but not Sgo1, specifically interacts with Mad2 in a manner that strongly resembles the interactions of Mad2 with Mad1 or Cdc20. Sgo2 contains a Mad1/Cdc20-like Mad2-interaction motif and competes with Mad1 and Cdc20 for binding to Mad2. NMR and biochemical analyses show that shugoshin binding induces similar conformational changes in Mad2 as do Mad1 or Cdc20. Mad2 binding regulates fine-tuning of Sgo2's sub-centromeric localization. Mad2 binding is conserved in the only known Xenopus laevis shugoshin homologue and, compatible with a putative meiotic function, the interaction occurs in oocytes.     

Development of a simple PCR-based assay for the identification of triazine resistance in the noxious plant common ragweed (Ambrosia artemisiifolia) and its applicability in higher plants

Bidirectional allele-specific PCR (Bi-PASA) was used to detect a point mutation causing triazine resistance in common ragweed (Ambrosia artemisiifolia). The external primers amplified a 278 bp standard DNA fragment in all genotypes. In the susceptible S264S genotypes, a 208 bp fragment was expected while in resistant S264G common ragweed genotypes a 109 bp band was expected. In resistant plants, both the wild and mutant type fragments were detected, indicating that the original triazine sensitive cpDNA is maintained in a heteroplasmic state in the resistant S264G genotypes. Additionally, in silico analysis confirmed the potential applicability of our diagnostic assay for other plant species. In 24 out of 74 taxa (32%), the assay could be used without any change, while in the others some of the primers should be redesigned before use.     

Redistribution of CB1 cannabinoid receptors in the acute and chronic phases of pilocarpine-induced epilepsy

The endocannabinoid system plays a central role in retrograde synaptic communication and may control the spread of activity in an epileptic network. Using the pilocarpine model of temporal lobe epilepsy we examined the expression pattern of the Type 1 cannabinoid receptor (CB1-R) in the hippocampi of CD1 mice at survival times of 2 hours, 1 day, 3 days and 2 months (acute, latent and chronic phases). Based on the behavioral signs of the acute seizures, animals were classified as "weakly" or "strongly" epileptic using the modified Racine scale. Mice of the weak group had mild seizures, whereas seizures in the strong group were frequent with intense motor symptoms and the majority of these animals developed sclerosis in the chronic phase. In control samples the most intense staining of CB1-R-positive fibers was found in the molecular layer of the dentate gyrus and in str. pyramidale of the cornu Ammonis. In weak animals no significant changes were seen at any survival time compared to controls. In strong animals, however, in the acute phase, a massive reduction in CB1-R-stained terminals occurred in the hippocampus. In the latent phase CB1-R immunoreactivity gradually recovered. In the chronic phase, CB1-immunostaining in sclerotic samples was stronger throughout the hippocampus. Quantitative electron microscopic analysis showed an increase in the number of CB1-R-positive terminals in the dentate gyrus. Moreover, the number of immunogold particles significantly increased in GABAergic terminals. Our results suggest a proconvulsive downregulation of CB1 receptors in the acute phase most probably due to receptor internalization, followed by compensatory upregulation and sprouting in the chronic phase of epilepsy. In conclusion, the changes in CB1 receptor expression pattern revealed in this study are associated with the severity of hippocampal injury initiated by acute seizures that ultimately leads to sclerosis in the vulnerable regions in the chronic phase.     

Genetic variability of Xrcc3 and Rad51 modulates the risk of head and neck cancer

A case-control study was conducted to analyze the possible associations between the head and neck cancer (HNC) risk and fourteen single nucleotide polymorphisms (SNPs) and haplotypes in Xrcc3 and Rad51 genes. This study involved 81 HNC cases and 111 healthy control subjects. A significant risk-increasing effect of rs3212057 (p.Arg94His) SNP in Xrcc3 (OR=6.6; p<0.01) was observed. On the other hand, risk-decreasing effect was found for rs5030789 (g.3997A>G) and rs1801321 (c.-60G>T) in 5' near gene and 5'UTR regions of Rad51, respectively (OR=0.3 and OR=0.2, p<0.05, respectively). Moreover, these effects were shown to be modulated by tobacco-smoking status and gene-gene interactions. Concluding, the genetic variability of Xrcc3 and/or Rad51 genes might be of relevance with respect to HNC risk.     

Macrofungal diversity in disturbed vegetation types in North-East Hungary

Macrofungi play an extraordinarily important role in the catalysis of the nutrient cycle of deciduous and coniferous forests. Habitat degradation adversely influences the number of fruiting bodies of macrofungi and diminishes the diversity of the fungal community. The diversity of the terricolous- and lignicolous macrofungi assemblages were compared in stands of semi-natural and two plant associations modified by humans in different degrees in North-East Hungary. We used data from 15 permanent plots that were sampled for vascular plants and macrofungi. Rank-abundance curves and Rényi’s diversity profiles were applied for diversity research. The results indicated that structure and diversity of the terricolous macrofungi assemblages were mainly influenced by climatic and habitat conditions and the degradation of the plant associations to a lesser degree. The diversity of lignicolous macrofungi was primarily affected by the continuous presence, quality, and quantity of deadwood. Accordingly, the form and degree of forest management, as well as the age of the growing stocks, influenced community structure. If nature conservation planning and conservation activity are based on those biodiversity indicators which are good proxies for macrofungi biodiversity, the latter might be optimal for preserving macrofungi biodiversity.     

Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.     

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs^∗57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.     

Functioning of the TA cassette of streptococcal plasmid pSM19035 in various Gram-positive bacteria

Toxin-antitoxin (TA) systems are common in microorganisms and are frequently found in the chromosomes and low-copy number plasmids of bacterial pathogens. One such system is carried by the low copy number plasmid pSM19035 of the pathogenic bacterium Streptococcus pyogenes. This plasmid encodes an omega-epsilon-zeta cassette that ensures its stable maintenance by post-segregational killing of plasmid-free cells. In this study, the activity of the ω-ε-ζ cassette was examined in various Gram-positive bacteria with a low G/C content in their DNA. The broad host range of pSM19035 was confirmed and the copy number of a truncated derivative in transformed strains was determined by real-time qPCR.