Long-term field study on stabilization of contaminated wastes by growing clonally reproduced Silene vulgaris calamine ecotype

Plant and Soil - Pseudo-metallophyte Silene vulgaris frequently colonizes polluted areas. We investigated whether plants obtained under in vitro conditions can be used to form long-term communities...     

Relationships between the properties of Spitsbergen soil,number and biodiversity of rhizosphere microorganisms,and heavy metal concentration in selected plant species

Plant and Soil - Ethylene-insensitive mutation (ein)-conferred Arabidopsis tolerance to Cd has been reported. However, the mechanisms involved are far from clear. This study explores possible...     

Pertactin-deficient Bordetella pertussis isolates in Poland—a country with whole-cell pertussis primary vaccination

The introduction of pertussis vaccination in the 1950s resulted in a significant decrease in the incidence of disease. However, since the 1990s many highly vaccinated countries have observed the re-emergence of the disease. One of the causes of this phenomenon might be related to the adaptation of Bordetella pertussis to vaccination. The purpose of the presented study was an investigation of the emergence and spread of vaccine antigen-deficient B. pertussis isolates in Poland and genomic characterization of the currently circulating pathogen population using PFGE, MLVA and MAST. The results revealed that all tested isolates expressed Ptx, FHA and ACT antigens but 15.4% (4/26) of isolates from 2010 to 2016 were Prn-deficient. Moreover, one TcfA-deficient isolate was collected in 2015. The genotyping showed a genetic distinction between the isolates circulating in 2010–2016 and isolates from previous periods. The majority of currently circulating isolates belonged to PFGE group IV (96.2%), type MT27 (73.1%), and carried ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-1 alleles (61.5%). The unique genetic structure of the B. pertussis population in Poland has changed since 2010 and became similar to that observed in countries with aP vaccination. This could be a result of increasing use of aP vaccines (60% of primary vaccination in 2013) over wP vaccines, which have been broadly used for primary vaccination in Poland for decades.     

Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry

This study aimed at establishing a sensitive multiple reaction monitoring‐mass spectrometry (MRM‐MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM‐MS analyses were performed using three transitions optimized on one purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM‐MS measurement were also determined. The method was applied to liver samples from ten non‐cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n = 12; PBC), primary sclerosing cholangitis (n = 10; PSC) or alcoholic liver disease (n = 12; ALD). The established method presented high sensitivity with limit of detection lower than 5 fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM‐based CYP3A4 protein quantification in homogenates and microsomes (r = 0.49, p < 0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM‐MS quantification of CYP3A4 in homogenates also correlated (r = 0.44; p < 0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion. The established method provides a sensitive tool to evaluate the CYP3A4 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury.     

Affilin Molecules Selected against the Human Papillomavirus E7 Protein Inhibit the Proliferation of Target Cells

Intracellular binding proteins can be applied as research tools for target validation and study of protein function in cells and potentially as therapeutics. The success of intracellular binding reagents depends on their affinity and specificity for target molecules, although their stability and functionality in the intracellular environment actually determine their usefulness for such application. Alternative binding proteins derived from scaffolds devoid of disulfide bonds are well suited for intracellular use, as their folding and stability are usually not impaired under reducing conditions. Here, we describe the generation of intracellular binding reagents called Affilin, based on the human γB-crystallin scaffold. The target was human papillomavirus E7 protein implicated in the development of cervical cancer. E7 binders were selected from the combinatorial γB-crystallin library by conventional phage display technique. Affilin variants specifically bound the E7 protein with affinities in the nanomolar range. Intracellular expression of Affilin molecules in E7-positive cells led to inhibition of cellular proliferation. The effect was specific, as the growth of E7-negative cells or cells expressing the wild-type γB-crystallin scaffold remained unaffected. These results demonstrate that the γB-crystallin scaffold allows the de novo generation of alternative binding proteins, which are suitable for intracellular applications as they retain their functionality in the reducing environment of mammalian cells.     

Molecular differences in the KRAS gene mutation between a primary tumor and related metastatic sites - case report and a literature review

In recent years the the set of diagnostic tools in colorectal cancers has been extended by the assessment of the KRAS gene status. Currently it is a necessary step in order to qualify patients for the targeted therapy. The results of the analysis of several studies revealed a high rate of compliance of the KRAS gene mutational status in primary and metastatic tumors. In this paper we present a rare case of incompatibility of the KRAS mutations in the primary tumor located in the colon and metastatic changes in the liver.     

The influence of abscisic acid on different polysomal populations in embryonal tissue during pea seeds germination

The influence of abscisic acid (ABA) on the processes of formation of different polysomal populations, their structures and stability in embryonal tissue during pea seeds germination was studied. The contents of total ribosomal fraction increased in all samples up to 72 h of germination and then decreased. The contents of polysomal population (FP, MBP, CBP and CMBP) extracted from the embryonal tissue after 72 hrs of germination of pea seeds were then quantified. It turned out that in examined tissue of control sample, fraction of free polysomes (FP) was the most abounded. This population of polysomes in sprouts decreased after ABA treatment. FP content decreased even more when the higher ABA concentration was applied during germination. Similar changes were observed in the fraction of membrane-bound polysomes (MBP). Quite different tendencies were found, however, in forming population of the cytoskeleton-membrane-bound polysomes (CMBP). The CMBP population content in embryonal tissue increased in a dosage dependent manner with increasing concentration of ABA applied during seed germination. This indicates the important role of CMBP fraction in synthesis of specific proteins in embryos in the time when processes of seeds germination are retarded by ABA. In the final part we examined the stability of polysomes isolated from sprouts of germinating seeds in water and sprouts isolated from seeds treated with ABA (100 μM) during germination. Total polysomes isolated from embryonal tissue of germinating seeds treated with ABA showed much higher resistance to exogenous ribonuclease digestion than total polysomes of control sample. The obtained results suggest that ABA influence on different polysomal population formation also controls their stability.     

Environment-dependent metabolic investments in the mixotrophic chrysophyte Ochromonas

Mixotrophic protists combine photosynthesis and phagotrophy to obtain energy and nutrients. Because mixotrophs can act as either primary producers or consumers, they have a complex role in marine food webs and biogeochemical cycles. Many mixotrophs are also phenotypically plastic and can adjust their metabolic investments in response to resource availability. Thus, a single species's ecological role may vary with environmental conditions. Here, we quantified how light and food availability impacted the growth rates, energy acquisition rates, and metabolic investment strategies of eight strains of the mixotrophic chrysophyte, Ochromonas. All eight Ochromonas strains photoacclimated by decreasing chlorophyll content as light intensity increased. Some strains were obligate phototrophs that required light for growth, while other strains showed stronger metabolic responses to prey availability. When prey availability was high, all eight strains exhibited accelerated growth rates and decreased their investments in both photosynthesis and phagotrophy. Photosynthesis and phagotrophy generally produced additive benefits: In low-prey environments, Ochromonas growth rates increased to maximum, light-saturated rates with increasing light but increased further with the addition of abundant bacterial prey. The additive benefits observed between photosynthesis and phagotrophy in Ochromonas suggest that the two metabolic modes provide nonsubstitutable resources, which may explain why a tradeoff between phagotrophic and phototrophic investments emerged in some but not all strains.     

DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.