Non-empirical quantum chemical studies on electron transfer reactions in trans- and cis-diamminedichloroplatinum(II) complexes

The search was made for theoretical confirmation of hypothesis that mechanism of cisplatin cytotoxicity is based on dissociative electron transfer (ET) processes. Applying quantum chemical calculations based on supermolecular approach, the reactions mimicking presumed steps of cisplatin activation were evaluated. The electronic structure of model systems: cis- and transplatin with free electrons, hydrated electrons, and water, was studied by using density functional (DFT) within the Huzinaga basis set and GAUSSIAN-09 package. The respective energy was evaluated with the use of B3LYP density hybrid functional. The calculations were performed for gas phase and water solution; the solvent effects were studied by using the polarizable continuum model. Analysis of the energetic and structural parameters of cisplatin vs. transplatin behavior in the model systems leads to conclusion: there are two possible ways of cisplatin biotransformation, hydrolysis and hydrated electron impact, dependent on the medium redox state.     

Accuracy of a remote quantitative image analysis in the whole slide images

The rationale for choosing a remote quantitative method supporting a diagnostic decision requires some empirical studies and knowledge on scenarios including valid telepathology standards. The tumours of the central nervous system [CNS] are graded on the base of the morphological features and the Ki-67 labelling Index [Ki-67 LI]. Various methods have been applied for Ki-67 LI estimation. Recently we have introduced the Computerized Analysis of Medical Images [CAMI] software for an automated Ki-67 LI counting in the digital images. Aims of our study was to explore the accuracy and reliability of a remote assessment of Ki-67 LI with CAMI software applied to the whole slide images [WSI]. The WSI representing CNS tumours: 18 meningiomas and 10 oligodendrogliomas were stored on the server of the Warsaw University of Technology. The digital copies of entire glass slides were created automatically by the Aperio ScanScope CS with objective 20x or 40x. Aperio's Image Scope software provided functionality for a remote viewing of WSI. The Ki-67 LI assessment was carried on within 2 out of 20 selected fields of view (objective 40x) representing the highest labelling areas in each WSI. The Ki-67 LI counting was performed by 3 various methods: 1) the manual reading in the light microscope - LM, 2) the automated counting with CAMI software on the digital images - DI , and 3) the remote quantitation on the WSIs - as WSI method. The quality of WSIs and technical efficiency of the on-line system were analysed. The comparative statistical analysis was performed for the results obtained by 3 methods of Ki-67 LI counting. The preliminary analysis showed that in 18% of WSI the results of Ki-67 LI differed from those obtained in other 2 methods of counting when the quality of the glass slides was below the standard range. The results of our investigations indicate that the remote automated Ki-67 LI analysis performed with the CAMI algorithm on the whole slide images of meningiomas and oligodendrogliomas could be successfully used as an alternative method to the manual reading as well as to the digital images quantitation with CAMI software. According to our observation a need of a remote supervision/consultation and training for the effective use of remote quantitative analysis of WSI is necessary.     

Nonlinear changes in the activity of the oxygen-dependent demethylase system in Rhodococcus erythropolis cells in the presence of low and very low doses of formaldehyde

ABSTRACT: The effect of exogenous, highly diluted formaldehyde on the rate of demethylation/re-methylation of veratric acid by the bacteria Rhodococcus erythropolis was studied using electrophoretic and microscopic techniques. The activity of 4-O-demethylase, responsible for accumulation of vanillic acid, and the levels of veratric and vanillic acids were determined using capillary electrophoresis. Formaldehyde was serially diluted at 1:100 ratios, and the total number of iterations was 20. After incubation of the successive dilutions of formaldehyde with the bacteria, demethylase activity oscillated in a sinusoidal manner. It was established using capillary electrophoresis that methylation of vanillic acid to veratric acid occurred at a double rate, as shown by the doubled fluctuation in the concentration of veratrate. There were also changes in the NADH oxidase activity, which is associated with methylation processes. Microscopic observations revealed the presence of numerous enlarged vacuoles in bacterial cells during the accumulation of large amounts of vanillic acid, and their disappearance together with a decrease in 4-O-demethylase activity. The presented results give evidence for the ability of living cells to detect the presence of submolecular concentrations of biological effectors in their environment and provide a basis for a scientific explanation of the law of hormesis and the therapeutic effect of homeopathic dilutions.     

NFX1-LIKE2 (NFXL2) suppresses abscisic acid accumulation and stomatal closure in Arabidopsis thaliana

The NFX1-LIKE1 (NFXL1) and NFXL2 genes were identified as regulators of salt stress responses. The NFXL1 protein is a nuclear factor that positively affects adaptation to salt stress. The nfxl1-1 loss-of-function mutant displayed reduced survival rates under salt and high light stress. In contrast, the nfxl2-1 mutant, defective in the NFXL2 gene, and NFXL2-antisense plants exhibited enhanced survival under these conditions. We show here that the loss of NFXL2 function results in abscisic acid (ABA) overaccumulation, reduced stomatal conductance, and enhanced survival under drought stress. The nfxl2-1 mutant displayed reduced stomatal aperture under all conditions tested. Fusicoccin treatment, exposition to increasing light intensities, and supply of decreasing CO(2) concentrations demonstrated full opening capacity of nfxl2-1 stomata. Reduced stomatal opening presumably is a consequence of elevated ABA levels. Furthermore, seedling growth, root growth, and stomatal closure were hypersensitive to exogenous ABA. The enhanced ABA responses may contribute to the improved drought stress resistance of the mutant. Three NFXL2 splice variants were cloned and named NFXL2-78, NFXL2-97, and NFXL2-100 according to the molecular weight of the putative proteins. Translational fusions to the green fluorescent protein suggest nuclear localisation of the NFXL2 proteins. Stable expression of the NFXL2-78 splice variant in nfxl2-1 plants largely complemented the mutant phenotype. Our data show that NFXL2 controls ABA levels and suppresses ABA responses. NFXL2 may prevent unnecessary and costly stress adaptation under favourable conditions.     

Polymorphisms of angiotensin II type 1 receptor gene and those of angiotensinogen point at culprit artery in ST-segment elevation myocardial infarction

The aim of the study was to determine whether the presence of angiotensin II type 1 receptor 1166A/C gene polymorphism and two polymorphisms of angiotensinogen, namely Met235Thr and Thr174Met, pointed at the culprit artery in patients with ST-segment elevation myocardial infarction (STEMI).     

G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase

The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL), which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1 switch gene-2 (G0S2). CGI-58 activates and G0S2 inhibits ATGL activity. In contrast to mice, the functional role of G0S2 in human adipocyte lipolysis is poorly characterized. Here we show that overexpression or silencing of G0S2 in human SGBS adipocytes decreases and increases lipolysis, respectively. Human G0S2 is upregulated during adipocyte differentiation and inhibits ATGL activity in a dose-dependent manner. Interestingly, C-terminally truncated ATGL mutants, which fail to localize to lipid droplets, translocate to the lipid droplet upon coexpression with G0S2, suggesting that G0S2 anchors ATGL to lipid droplets independent of ATGL''s C-terminal lipid binding domain. Taken together, our results indicate that G0S2 also regulates human lipolysis by affecting enzyme activity and intracellular localization of ATGL. Increased lipolysis is known to contribute to the pathogenesis of insulin resistance, and G0S2 expression has been shown to be reduced in poorly controlled type 2 diabetic patients. Our data indicate that downregulation of G0S2 in adipose tissue could represent one of the underlying causes leading to increased lipolysis in the insulin-resistant state.     

Diversity of integron- and culture-associated antibiotic resistance genes in freshwater floc

Clinically important antibiotic resistance genes were detected in culturable bacteria and class 1 integron gene cassettes recovered from suspended floc, a significant aquatic repository for microorganisms and trace elements, across freshwater systems variably impacted by anthropogenic activities. Antibiotic resistance gene cassettes in floc total community DNA differed appreciably in number and type from genes detected in bacteria cultured from floc. The number of floc antibiotic resistance gene cassette types detected across sites was positively correlated with total (the sum of Ag, As, Cu, and Pb) trace element concentrations in aqueous solution and in a component of floc readily accessible to bacteria. In particular, concentrations of Cu and Pb in the floc component were positively correlated with floc resistance gene cassette diversity. Collectively, these results identify suspended floc as an important reservoir, distinct from bulk water and bed sediment, for antibiotic resistance in aquatic environments ranging from heavily impacted urban sites to remote areas of nature reserves and indicate that trace elements, particularly Cu and Pb, are geochemical markers of resistance diversity in this environmental reservoir. The increase in contamination of global water supplies suggests that aquatic environments will become an even more important reservoir of clinically important antibiotic resistance in the future.     

Cohesin rings devoid of scc3 and pds5 maintain their stable association with the DNA

Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion in vivo using degron tagging in budding yeast. The previously described DHFR-based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 "core" subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.