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Anthracyclines use is limited by profound cardiotoxicity. Involvement of miRNAs in anthracycline-induced cardiotoxicity (AIC) is still not completely understood. Thus, the expression of AIC-related microRNAs was determined in rat atria and ventricles after doxorubicin (DOX) and liposomal doxorubicin (L-DOX) administration. Vehiculum, DOX or L-DOX were applied intraperitoneally in a single dose to male Wistar rats (3 groups: control, DOX and L-DOX, respectively). Rats were sacrificed after 24 h, and samples from left atrium (LA)/ventricle (LV) and right atrium (RA)/ventricle (RV) were obtained. Expressions of miR-208a, let-7g and snU6 were determined using qRT-PCR. In the control group, miR-208a was highly abundant in the atria compared to the ventricles and in the left-sided structures compared to the right-sided structures, while let-7g showed only atrio-ventricular gradient with predominant expression in the atria. Administration of both DOX and L-DOX resulted in 38.87 and 23.57% reduction in miR-208a expression in the LV (p = 0.028) and in 13.79 and 14.70% reduction in let-7g expression in the LA (p = 0.015), respectively. Acute administration of DOX/L-DOX alters expression of miR-208a in LV and of let-7g in LA. These changes may partly contribute to the development of AIC.

Background: Failure in intracellular zinc accumulation is a key process in prostate carcinogenesis. Nevertheless, epidemiological studies of zinc administration have provided contradicting results. In order to examine the impact of the artificial intracellular increase of zinc(II) ions on prostate cancer metabolism, PNT1A, 22Rv1, and PC-3 prostatic cell lines-depicting different stages of cancer progression-and their zinc-resistant counterparts were used. To determine "benign" and "malignant" metabolic profiles, amino acid patterns, gene expression, and antioxidant capacity of these cell lines were assessed., Methods: Amino acid profiles were examined using an ion-exchange liquid chromatography. Intracellular zinc content was measured by atomic absorption spectrometry. Metallothionein was quantified using differential pulse voltammetry. The content of reduced glutathione was determined using high performance liquid chromatography coupled with an electrochemical detector. Cellular antioxidant capacity was determined by the ABTS test and gene expression analysis was performed by qRT-PCR., Results and Conclusions: Long-term zinc treatment was shown to reroute cell metabolism from benign to more malignant type. Long-term application of high concentration of zinc(II) significantly enhanced cisplatin resistance, invasiveness, cellular antioxidant capacity, synthesis of glutathione, and expression of treatment resistance- and stemness-associated genes (SOX2, POU5F1, BIRC5). Tumorous cell lines universally displayed high accumulation of aspartate and sarcosine and depletion of essential amino acids. Increased aspartate/threonine, aspartate/methionine, and sarcosine/serine ratios were associated with cancer phenotype with high levels of sensitivity and specificity. Prostate 77: 604-616, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10 -5 , comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10 -5 ), if sufficient cells (>4 × 10 6 , preferably more) are evaluated., (© 2017 by The American Society of Hematology.)

Background: Anthracycline antibiotic doxorubicin (DOX) is a very potent and extensively prescribed chemotherapeutic drug. It is widely utilized in the therapy of variety of haematological and solid tumours, although its administration is commonly accompanied with several severe side effects. The most serious one is a development of dose-dependent and cumulative cardiotoxicity. In the course of time, many strategies have been investigated in order to avoid or at least to diminish DOX-induced cardiac dysfunction; these include reduction of toxic effect by coadministration with iron chelators (dexrazoxane), trastuzumab, taxanes, statins, and ACE-inhibitors. However, the attenuation of cardiotoxic effect is still not satisfactory yet., Objective: This review represents an overall appraisal of studies concerning with the utilization of various doxorubicinloaded nanoparticles in the cancer treatment with specific emphasis on those studies evaluating their influence on the reduction of heart tissue damage., Conclusion: Introduction of nanoscale drug delivery systems undoubtedly represents nowadays one of the most promising tools for lowering systemic toxicity. Nanoparticles enable to target the therapeutic payload directly towards the tumor tissue, thus leading to the increased accumulation of the drug in the desired tissue and simultaneously protecting surrounding healthy tissues., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The genus Rickettsia (Rickettsiales: Rickettsiaceae) includes Gram-negative, small, obligate intracellular, nonmotile, pleomorphic coccobacilli bacteria transmitted by arthropods. Some of them cause human and probably also animal disease (life threatening in some patients). In these guidelines, we give clinical practice advices (microscopy, serology, molecular tools, and culture) for the microbiological study of these microorganisms in clinical samples. Since in our environment rickettsioses are mainly transmitted by ticks, practical information for the identification of these arthropods and for the study of Rickettsia infections in ticks has also been added.

Histopathological analysis can provide important information in long-term experiments with total artificial heart (TAH). Recently, a new type of blood pump, the helical flow total artificial heart (HF-TAH) was developed. This study aimed to investigate the changes in selected vital organs in animal experiments with implanted HF-TAH. Samples from lung, liver, and kidneys from two female goats (No. 1301 and No. 1304) with implanted HF-TAH were analyzed. Tissue samples were fixed in 10% formaldehyde and 4 µm thick transverse sections were stained with hematoxylin-eosin (HE). Additional staining was done for detection of connective tissue (Masson-Goldner stain) and for detection of iron (hemosiderin) deposits (Perls stain). Sections were scanned at 100× and 500× magnification with a light microscope. Experiment no. 1301 survived 100 days (cause of termination was heavy damage of the right pump); experimental goat no.1304 survived 68 days and was sacrificed due to severe right hydrodynamic bearing malfunction. Histopathological analysis of liver samples proved signs of chronic venostasis with limited focal necrotic zones. Dilated tubules, proteinaceous material in tubular lumen, and hemosiderin deposits were detected in kidney samples. Contamination of the organs by embolized micro-particles was suspected at the autopsy after discovery of visible damage (scratches) of the pump impeller surface (made from titanium alloy) in both experiments. Sporadic deposits of foreign micro-particles (presumably titanium) were observed in most of the analyzed parenchymal organs. However, the described deposits were not in direct connection with inflammatory reactions in the analyzed tissues. Histopathological analysis showed the presence of minimal contamination of the lung, kidney, and liver tissue samples by foreign material (titanium very likely). The analysis showed only limited pathological changes, especially in liver and kidneys, which might be attributed to the influence of artificial perfusion often observed in chronic TAH experiments., (© 2016 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation.)

In this study we show that anti-tumor effect of sulforaphane (SFN) is partially realized through the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). This effect was verified in vitro on three different stable cell lines and also in vivo on the model of nude mice with developed tumors. Early response (6 hours) of A2780 ovarian carcinoma cells to SFN treatment involves generation of mitochondrial ROS and increased transcription of NRF2 and its downstream regulated genes including heme oxygenase 1, NAD(P)H:quinine oxidoreductase 1, and KLF9. Prolonged SFN treatment (24 hours) upregulated expression of NRF2 and IP3R1. SFN induces a time-dependent phosphorylation wave of HSP27. Use of IP3R inhibitor Xestospongin C (Xest) attenuates both SFN-induced apoptosis and the level of NRF2 protein expression. In addition, Xest partially attenuates anti-tumor effect of SFN in vivo. SFN-induced apoptosis is completely inhibited by silencing of IP3R1 gene but only partially blocked by silencing of NRF2; silencing of IP3R2 and IP3R3 had no effect on these cells. Xest inhibitor does not significantly modify SFN-induced increase in the rapid activity of ARE and AP1 responsive elements. We found that Xest effectively reverses the SFN-dependent increase of nuclear content and decrease of reticular calcium content. In addition, immunofluorescent staining with IP3R1 antibody revealed that SFN treatment induces translocation of IP3R1 to the nucleus. Our results clearly show that IP3R1 is involved in SFN-induced apoptosis through the depletion of reticular calcium and modulation of transcription factors through nuclear calcium up-regulation., Competing Interests: The authors declare that they have no conflicts of interest.

Background: Recently, a new rickettsia named 'Candidatus Rickettsia vini' belonging to the spotted fever group has been molecularly detected in Ixodes arboricola ticks in Spain, the Czech Republic, Slovakia and Turkey, with prevalence reaching up to 100 %. The aim of this study was to isolate this rickettsia in pure culture, and to describe it as a new Rickettsia species., Methods: A total of 148 ornitophilic nidicolous ticks Ixodes arboricola were collected in a forest near Breclav (Czech Republic) and examined for rickettsiae. Shell vial technique was applied to isolate rickettsiae in Vero cells. Rickettsial isolation was confirmed by optical microscopy and sequencing of partial sequences of the rickettsial genes gltA, ompA, ompB, and htrA. Laboratory guinea pigs and chickens were used for experimental infestations and infections. Animal blood sera were tested by immunofluorescence assay employing crude antigens of various rickettsiae., Results: Rickettsia vini n. sp. was successfully isolated from three males of I. arboricola. Phylogenetic analysis of fragments of 1092, 590, 800, and 497 nucleotides of the gltA, ompA, ompB, and htrA genes, respectively, showed closest proximity of R. vini n. sp. to Rickettsia japonica and Rickettsia heilongjiangensis belonging to the spotted fever group. Experimental infection of guinea pigs and chickens with R. vini led to various levels of cross-reactions of R. vini-homologous antibodies with Rickettsia rickettsii, Rickettsia parkeri, 'Candidatus Rickettsia amblyommii', Rickettsia rhipicephali, Rickettsia bellii, and Rickettsia felis. Laboratory infestations by R. vini-infected I. arboricola larvae on chickens led to no seroconversion to R. vini n. sp., nor cross-reactions with R. rickettsii, R. parkeri, 'Ca. R. amblyommii', R. rhipicephali, R. bellii or R. felis., Conclusions: Our results suggest that R. vini n. sp. is possibly a tick endosymbiont, not pathogenic for guinea pigs and chickens. Regarding specific phenotypic characters and significant differences of DNA sequences in comparison to the most closely related species (R. japonica and R. heilongjiangensis), we propose to classify the isolate as a new species, Rickettsia vini.

A novel rickettsial sequence in the citrate synthase gltA gene indicating a novel Rickettsia species has been detected in 7 out of 4524 Ixodes ricinus ticks examined within several surveys performed in the Czech Republic from 2005 to 2009. This new Candidatus Rickettsia sp. sequence has been found in 2 nymphs feeding on wild birds (Luscinia megarhynchos and Erithacus rubecula), in a male tick from vegetation, and 4 ticks feeding on a dog (3 males, 1 female tick). Portions of the ompA, ompB, sca4, and htrA genes were not amplifiable in these samples. A maximum likelihood tree of rickettsiae based on comparisons of partial amino acid sequences of citrate synthase and nucleotide sequences of 16S rDNA genes and phylogenetic analysis revealed a basal position of the novel species in the proximity of R. bellii and R. canadensis. The novel species has been named 'Candidatus Rickettsia mendelii' after the founder of genetics, Gregor Mendel., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Complex karyotypes are seen in approximately 20% of patients with myelodysplastic syndromes (MDS) and are associated with a high risk of transformation to acute myeloid leukemia and poor outcomes in patients. Copy number neutral loss of heterozygosity (CN-LOH, i.e., both copies of a chromosomal pair or their parts originate from one parent) might contribute to increased genomic instability in the bone-marrow cells of patients with MDS. The pathological potential of CN-LOH, which arises as a clonal aberration in a proportion of somatic cells, consists of tumor suppressor gene and oncogene homozygous mutations. The aim of our study was to evaluate the frequency of CN-LOH at 17p in bone-marrow cells of newly diagnosed MDS patients with complex chromosomal aberrations and to assess its correlation with mutations in the TP53 gene (17p13.1). CN-LOH was detected in 40 chromosomal regions in 21 (29%) of 72 patients analyzed. The changes in 27 of the 40 regions identified were sporadic. The most common finding was CN-LOH of the short arm of chromosome 17, which was detected in 13 (18%) of 72 patients. A mutational analysis confirmed the homozygous mutation of TP53 in all CN-LOH 17p patients, among which two frameshift mutations are not registered in the International Agency for Research on Cancer TP53 Database. CN-LOH 17p correlated with aggressive disease (median overall survival 4 months) and was strongly associated with a complex karyotype in the cohort studied, which might cause rapid disease progression in high-risk MDS. No other CN-LOH region previously recorded in MDS or AML patients (1p, 4q, 7q, 11q, 13q, 19q, 21q) was detected in our cohort of patients with complex karyotype examined at the diagnosis of MDS. The LOH region appeared to be balanced (i.e., with no DNA copy number change) when examined with conventional and molecular cytogenetic methods. Therefore, a microarray that detects single-nucleotide polymorphisms is an ideal method with which to identify and further characterize CN-LOH. Our data should specify the prognosis and should lead to the identification of potential targets for therapeutic interventions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Calcium plays a crucial role in numerous processes in living systems, from both intracellular and intercellular signalling to blood clotting. Calcium can be replaced by strontium in various intracellular processes due to high level of their similarity and strontium thus may serve as a valuable tool for different experimental studies. On the other hand, strontium is also used in clinical medicine and is commonly taken to the human body with food and water. The negative cardiac side effects of strontium therapy of osteoporosis and bone metastases are well known, but still not fully explained. This fact explains enhanced interest in this element and its impact on human body. This article reviews effects of calcium and strontium on several biochemical and physiological processes, with special emphasis on cardiac muscle.

Ticks were collected from captive reptiles, wild birds, and incidentally from humans at two locations in Honduras and part of these were tested for the presence of Rickettsia using polymerase chain reaction. The following species of ticks were found: Amblyomma dissimile on Iguanidae reptiles, Amblyomma longirostre and Amblyomma nodosum on birds, and Amblyomma mixtum (Amblyomma cajennense complex) on humans. A. dissimile was infected with Rickettsia sp. strain Colombianensi. Both A. longirostre and A. mixtum were infected with Candidatus 'Rickettsia amblyommii'. This study provides the first report of rickettsial infections in ticks from reptiles, birds and humans in Honduras. New host - Amblyomma tick associations are documented., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Isolated heart model is a relevant tool for cardiovascular system studies. It represents a highly reproducible model for studying broad spectrum of biochemical, physiological, morphological, and pharmaceutical parameters, including analysis of intrinsic heart mechanics, metabolism, and coronary vascular response. Results obtained in this model are under no influence of other organ systems, plasma concentration of hormones or ions and influence of autonomic nervous system. The review describes various isolated heart models, the modes of heart perfusion, and advantages and limitations of various experimental setups. It reports the improvements of perfusion setup according to Langendorff introduced by the authors.

Haloperidol is a neuroleptic drug used for a medication of various psychoses and deliria. Its administration is frequently accompanied by cardiovascular side effects, expressed as QT interval prolongation and occurrence of even lethal arrhythmias. Despite these side effects, haloperidol is still prescribed in Europe in clinical practice. Haloperidol binds to sigma receptors that are coupled with inositol 1,4,5-trisphosphate (IP3) receptors. Sigma receptors are expressed in various tissues, including heart muscle, and they modulate potassium channels. Together with IP3 receptors, sigma receptors are also involved in calcium handling in various tissues. Therefore, the present work aimed to study the effects of long-term haloperidol administration on the cardiac function. Haloperidol (2 mg/kg once a day) or vehiculum was administered by intraperitoneal injection to guinea pigs for 21 consecutive days. We measured the responsiveness of the hearts isolated from the haloperidol-treated animals to additional application of haloperidol. Expression of the sigma 1 receptor and IP3 receptors was studied by real time-PCR and immunohistochemical analyses. Haloperidol treatment caused the significant decrease in the relative heart rate and the prolongation of QT interval of the isolated hearts from the haloperidol-treated animals, compared to the hearts isolated from control animals. The expression of sigma 1 and IP3 type 1 and type 2 receptors was increased in both atria of the haloperidol-treated animals but not in ventricles. The modulation of sigma 1 and IP3 receptors may lead to altered calcium handling in cardiomyocytes and thus contribute to changed sensitivity of cardiac cells to arrhythmias.

The aim of this study was to analyze the prevalence of rickettsiae in the tree-hole tick Ixodes arboricola in the Czech Republic and Slovakia. During May to September of 2009 and 2013, bird boxes belonging to three different areas were screened for ticks. In total, 454 nestlings and 109 nests of 10 hole-breeding bird species were examined. Ticks were found on Ficedula albicollis, Parus major, Cyanistes caeruleus and Sitta europaea and/or in their nests. In total, 166 ticks (17 nymphs, 10 males and 139 females) were found at 3 areas (arithmetic mean±standard error: 55.3±45.9). All ticks were tested for the presence of Rickettsia species by polymerase chain reaction targeting the rickettsial genes gltA, ompA, ompB and htrA and amplicon sequencing. All individuals except 3 nymphs were infected with 'Candidatus Rickettsia vini'. Multilocus sequence typing showed closest proximity to Rickettsia japonica and Rickettsia heilongjiangensis cluster. The presence of 'Ca. R. vini' is reported for the first time in Slovakia., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry., (© 2014 International Society for Advancement of Cytometry.)

Aim: The cardio-ankle vascular index (CAVI) is a sensitive non-invasive marker of arterial stiffness and atherosclerosis. The aim of this work was to compare the CAVI values in patients with dyslipidemia (without diabetes mellitus and hypertension) and healthy controls., Methods: A Total 248 subjects with dyslipidemia (104 men, 144 women), 55.0 (95% CI 30-70) years of age with combined hyperlipidemia or primary hypercholesterolemia and 537 healthy controls (244 men, 293 women) 40.0 (95% CI 26-62) years of age were included in this study. Fasting blood samples were collected to measure the serum total cholesterol, triglyceride, HDL-cholesterol and apolipoprotein A1 and B levels. The LDL cholesterol level was also calculated, and the CAVI was measured using the VaSera(®) 1500 system., Results: The CAVI values were significantly higher in the dyslipidemic patients (8.08, 95% CI 6.00-10.05) than in the controls (7.11, 95% CI 5.77-9.05; p < 0.01). In addition, the CAVI values were elevated in both subgroups of patients with hypercholesterolemia (7.95, 95% CI 5.85-6.90; p < 0.01) and combined hyperlipidemia (8.30, 95% CI 6.60-10.15; p < 0.01) in comparison with those observed in the controls. After adopting the propensity score method in order to balance the confounding factors (age, gender, body mass index) and adjust the analysis for diastolic blood pressure, the CAVI values in the dyslipidemic patients remained significantly high (7.78, 95% CI 5.80-9.69) compared to that observed in the controls (7.31, 95% CI 5.44-9.35; p < 0.001). However, the CAVI values did not differ significantly between the controls and both subgroups of dyslipidemic patients(primary hypercholesterolemia, combined hyperlipidemia)., Conclusions: The present findings demonstrated that dyslipidemia increases the CAVI values in comparison to that seen in healthy subjects.

Polymorphic microsatellite loci were characterised for two louse species, the anopluran Polyplax serrata Burmeister, 1839, parasitising Eurasian field mice of the genus Apodemus Kaup, and the amblyceran Myrsidea nesomimi Palma et Price, 2010, found on mocking birds endemic to the Galápagos Islands. Evolutionary histories of the two parasites show complex patterns influenced both by their geographic distribution and through coevolution with their respective hosts, which renders them prospective evolutionary models. In P. serrata, 16 polymorphic loci were characterised and screened across 72 individuals from four European populations that belong to two sympatric mitochondrial lineages differing in their breadth of host-specificity. In M. nesomimi, 66 individuals from three island populations and two host species were genotyped for 15 polymorphic loci. The observed heterozygosity varied from 0.05 to 0.9 in P. serrata and from 0.0 to 0.96 in M. nesomimi. Deviations from the Hardy-Weinberg equilibrium were frequently observed in the populations of both parasites. Fst distances between tested populations correspond with previous phylogenetic data, suggesting the microsatellite loci are an informative resource for ecological and evolutionary studies of the two parasites.

Stress is a contributor of many cardiovascular diseases. Positive inotropic and chronotropic effects of catecholamines are regulated via β-adrenergic receptors (ARs). Many reports exist concerning changes of cardiac β1 - and β2 -ARs in stress, but only a few deal with modulation of cardiac β3 -AR. Our aim was to analyze the expression and binding sites of β1 -, β2 - and β3 -ARs and adenylyl cyclase activity in the left ventricle, and β3 -AR expression and binding in the left atrium of rats exposed to acute and chronic immobilization stress (IMO). The concentration of noradrenaline in the ventricle decreased, while adrenaline increased, especially after repeated IMO. The mRNA and protein levels, and binding sites of β3 -subtype significantly rose following chronic IMO, while all parameters for β2 -AR dropped after single and repeated exposure. Similarly, the mRNA levels and binding sites for β3 -subtype increased in the left atrium as a consequence of chronic IMO. The rise in β3 -subtypes and a drop in β2 -subtypes resulted in inhibition of adenylyl cyclase activity within the left ventricle. Taken together, among other factors, up-regulation of β3 -AR could represent an adaptation mechanism, which might be related to altered physiological function of the left ventricle and atrium during prolonged emotional stress and might serve cardioprotective function during catecholamine overload., (Copyright © 2013 John Wiley & Sons, Ltd.)

We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.

We report the use of a microfluidic microarray incorporating single molecule detection for the absolute quantification of protein copy number in solution. In this paper we demonstrate protocols which enable calibration free detection for two protein detection assays. An EGFP protein assay has a limit of detection of <30 EGFP proteins in a microfluidic analysis chamber (limited by non-specific background binding), with a measured limit of linearity of approximately 6 × 10(6) molecules of analyte in the analysis chamber and a dynamic range of >5 orders of magnitude in protein concentration. An antibody sandwich assay was used to detect unlabelled human tumour suppressor protein p53 with a limit of detection of approximately 21 p53 proteins and a dynamic range of >3 orders of magnitude. We show that these protocols can be used to calibrate data retrospectively to determine the absolute protein copy number at the single cell level in two human cancer cell lines.

MDS with complex chromosomal aberrations (CCA) are characterized by short survival and a high rate of transformation to AML. A comprehensive genome-wide analysis of bone-marrow cells of 157 adults with newly diagnosed MDS and CCA revealed a large spectrum of nonrandom genomic changes related to the advanced stages of MDS. Chromosome shattering, probably resulting from chromothripsis, was found in 47% of patients. Deleted chromosome 5 was unstable and often involved in different types of cryptic unbalanced rearrangements. No true monosomy 5 was observed. Patients with CCA involving deleted chromosome 5 had an extremely poor prognosis (median overall survival, 2 months)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The default mode network (DMN) decreases its activity when switching from a resting state to a cognitive task condition, while activity of the network engaged in the given task increases. Visual processing is typically disturbed in Parkinson's disease dementia (PDD)., Objective: Using functional MRI, we studied the DMN effective connectivity patterns in PDD as compared with cognitively normal patients with Parkinson's disease (PD) and healthy controls (HC) when switching from baseline to a visual cognitive task condition., Methods: In all, 14 PDD, 18 PD, and 18 age-matched healthy controls participated in this functional MRI study. We used a psychophysiological interaction analysis with the precuneus (PCu) as a seed. The threshold was set at p(FWE) <0.05., Results: The healthy controls showed greater PCu connectivity with the bilateral middle temporal/middle occipital gyri at baseline than during the task condition. The correlation direction changed from positive to negative. Both PD and PDD showed disturbed DMN connectivity with the brain regions that are involved in bottom-up visual processing. In PD, we also found impaired integration of the areas engaged in the ventral attentional network, which might reflect specific attentional deficits observed during the early course of PD. In mild PDD, we detected increased engagement of areas involved in the dorsal attentional network, which corresponds to increased top-down control in this patient group as compared to the healthy controls., Conclusion: Our results show impaired dynamic interplay between large scale brain networks in PD that spread far beyond the motor system.

The left and right ventricles fulfill different role in heart function. Here we compare chamber specific changes in local catecholamine concentrations; gene expression and the receptor protein amount of all three β-adrenoceptors (β-AR) in rat right heart ventricles exposed to acute (1 session) and repeated (7 sessions) immobilization stress (IMMO) vs. previously observed changes in left ventricles. Density of muscarinic receptors as main cardio-inhibitive receptors was also measured. In the right ventricles, noradrenaline and adrenaline were increased. No β1-AR changes were observed, in spite of the increased sympathetic activity. On the other hand, we have found a decrease of β2-AR gene expression (reduction to 30%) after 7 IMMO and protein (to 59%) after 1 IMMO. β3-AR gene expression was increased after 7 IMMO. Muscarinic receptor density was not changed. When comparing correlation in left and right ventricles, there was strong correlation between adrenaline and β2-AR gene expression, protein and β3-AR gene expression in the left ventricles while only correlation between adrenaline and β2-AR mRNA and protein in the right ventricles was found. Our results show that maintenance of cardiac homeostasis under stress conditions are to a great extent achieved by a balance between different receptors and also by a balanced receptor changes in left vs. right ventricles. Taken together, decrease of cardio-stimulating β2-AR represents a new important mechanism by which β2-AR contributes to the heart physiology.

Aim: Both aerobic training (AT) and electromyostimulation (EMS) of leg muscles improve exercise tolerance in patients suffering from chronic heart failure (CHF). It was speculated that combination of both methods might have an additive effect. This study was performed to evaluate the effects of a combination of AT and EMS in rehabilitation (RHB) of CHF patients., Patients and Methods: Patients (n=71; age 59 ± 10.2 yrs, NYHA II/III, EF 32 ± 7.1%) were randomized into 3 groups: a) group AT, b) group EMS, and c) group AT+EMS. AT protocol included standard activity on bicycle 3x a week at the level of individual anaerobic threshold. EMS (10 Hz, mode 20s "on"/20s "off") was applied to leg extensors for 2 h/day. Total time of given type of RHB was 12 weeks., Results: Data analysis revealed statistically significant improvements of patients in all experimental groups (averaged difference after 12 weeks of exercise as related to initial value: ∆VO2peak: +12.9%, ∆VO2AT: +9.3%, ∆Wpeak: +22.7%). No statistically significant difference among experimental groups was found. Quality of life (Minnesota Living with Heart Failure - MLHF) global score was significantly improved in all 3 groups: AT (∆MLHF: -27.9%; P=0.001), AT+EMS (∆MLHF: -29.1%; P=0.002), and EMS (∆MLHF: -16.6%; P=0.008). MLHF score in EMS group showed the smallest time-related improvement compared to AT and AT+EMS groups, and this difference in improvement between the groups was statistically significant (P=0.021)., Conclusion: No significant difference was found between the two types of exercise training.and nor did, their combination have any significant additional improvement.

Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene - enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified afterpurification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected. Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with nontransgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.

Haloperidol (HP) is used for the symptomatic treatment of psychosis, manic phases, hyperactivity, aggressiveness, and acute delirium. Long-term use leads to various adverse side effects, especially to severe impairment of extrapyramidal nerve tracts and in particular, altered QT interval and increased incidence of arrhytmias. It is believed that cytotoxicity of HP and its metabolites is responsible for both neurotoxicity and cardiotoxicity. Extrapyramidal and cardiac adverse side effects may be explained by the HP-induced oxidative stress, as implicated by many studies. HP was reported to induce lipid peroxidation with subsequent membrane changes, responsible for cell death. Vice versa, cells resistant to oxidative stress are also resistant to the toxic effects of HP. Similarly, high percentage of patients suffering from extrapyramidal symptoms treated by vitamin E and other lipid-soluble antioxidants demonstrates diminishing of these adverse side effects. HP's ability to induce oxidative stress by multi-modal action (increased metabolism of dopamine, decrease of glutathione content, induction of NF-κB transcription factor, and inhibition of complex I of respiratory chain) has been established just recently. This review brings summarizing view on the cytotoxicity of haloperidol and involvement of reactive oxygen species and oxidative stress HP-induced cytotoxicity.

Measuring protein expression in single cells is the basis of single cell proteomics. The sensitivity and dynamic range of a single cell immunoassay should ideally be such that proteins that are expressed between 1-10(6) copies per cell can be detected and counted. We have investigated the effect of miniaturizing antibody microarrays by reducing capture spot sizes from 100 μm to 15 μm using dip-pen nanolithography. We demonstrate that protocols developed for printing and passivating antibody capture spots using conventional pin-based contact printing can be directly transferred to dip-pen lithography whilst retaining the capture activity per unit area. Using a simple kinetic model, we highlight how the limit of detection and dynamic range of a sandwich immunoassay, respectively, increase and decrease when spot size is reduced. However, we show that reducing spot size is more effective than increasing assay chamber volume when seeking to multiplex such a microfluidic immunoassay. Although we make particular reference to single cell microfluidic immunoassays, the topics discussed here are applicable to capture assays in general.

Ischemic and reperfusion injury is a serious condition related to numerous biochemical and electrical abnormalities of the myocardium. It has been repeatedly studied in various animal models. In this study, the production of hydroxyl radicals and electrophysiological parameters were compared in three species. Rat, guinea pig and rabbit isolated hearts were perfused according to Langendorff under strictly identical conditions. The heart rate and arrhythmia were monitored during ischemia and reperfusion periods at defined time intervals; the production of hydroxyl radical was determined by HPLC as 2.5-dihydroxybenzoic acid (2.5-DHBA) formed by salicylic acid hydroxylation. Relationship between arrhythmias and production of 2.5-DHBA was studied. The inter-species differences were observed in timing of arrhythmias onset and their severity, and in the production of 2.5-DHBA in both ischemia and reperfusion. The most considerable changes were observed in rats, where arrhythmias appeared early and with highest severity during ischemia on one side and the regular rhythm was restored early and completely during reperfusion. The corresponding changes in the production of 2.5-DHBA were observed. It can be concluded that rat isolated heart is the most suitable model for evaluation of ischemia/reperfusion injury under given experimental conditions.

Objectives: Various reports have suggested that the involvement of normal-looking white matter with tumors is not limited to just signal abnormalities detectable on conventional imaging. Thus, the purpose of this study was to investigate the distant effects of glioblastomas and metastases on white matter using diffusion tensor imaging (DTI)., Materials and Methods: Data for 21 patients harboring a glioblastoma (n=12) or a metastasis (n=9) located at a distance of smaller or equal to 10mm from a DTI-based reconstruction of the pyramidal tract were analyzed, using regions of interest (ROIs) placed along the pyramidal tracts in the cerebral peduncle distant (>15 mm) from the tumor., Results: For the whole study population, fractional anisotropy (FA) was significantly lower on the side ipsilateral to the tumor (P<0.001), a difference that was also observed in the glioblastoma and metastasis subgroups. The trace value was significantly higher on the ipsilateral side in the whole population and metastasis subgroup, but not in the glioblastoma subgroup. The decrease in FA and the trace value increase were significant in a subgroup of patients with motor deficits, but not in those without such deficits., Conclusion: Hemispheric glioblastomas and metastases located close to the pyramidal tract induce diffusion changes in the tract that are observable at a distance of greater than 15 mm from the tumor border in the absence of T2 signal changes. These changes are different in glioblastomas and metastases, and mechanisms other than Wallerian degeneration may be contributing to the observed changes., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Glucocorticoids act via glucocorticoid receptors (GR), typically localized in the cytosol (cGR). Rapid action is probably mediated via membrane receptors (mGR). In corticotropin-releasing hormone knockouts (CRH-KO), basal plasma glucocorticoid levels do differ from wild type levels (WT), but are approximately ten times lower during exposure to immobilization stress (IMMO) in comparison to WT. We tested the following hypotheses: (1) the mice lung tissue GR basal numbers would not be changed in CRH-KO (because of similar glucocorticoid levels), (2) the number of GR would be changed in WT but not in KO during short (30, 90, and 120 min) IMMO (because of higher increase of glucocorticoid levels in WT). The basal levels of cGR were not changed in CRH-KO (compared to WT), while mGR were significantly lower (62 %) in CRH-KO. In WT, there was the only decrease (to 32 %) in cGR after 120 min when we also found an increase in mGR in WT (to 201 %). In CRH-KO, IMMO caused gradual decrease in cGR (to 52 % after 30 min, to 46 % after 90 min, and to 32 % after 120 min). In CRH-KO, the only increase in mGR appeared already at 30 min of IMMO. These data suggest, on the contrary to our hypotheses, that CRH-KO are more susceptible to GR changes in early phases of stress.

Mucosal immunization with non-living antigens usually requires the use of an adjuvant. The adjuvant activity of Bacillus firmus in the mucosal immunization of mice was described by our laboratory previously. In the present study, subcellular localization of B. firmus activities was followed. After mechanical disintegration, subcellular components of bacterium were fractionated by differential centrifugation and salting out. Bacterial cell walls, cytoplasmic membrane fraction, soluble cytoplasmic proteins, and ribosomal fractions were isolated. Their effect on the mouse immune system was studied. Lymphocyte proliferation and immunoglobulin formation in vitro were stimulated by bacterial cell wall (BCW), cytoplasmic membrane (CMF), and ribosomal fractions. BCW and CMF increased antibody formation after intratracheal immunization of mice with influenza A and B viruses, and increased protection against subsequent infection with influenza virus. The BCW fraction even induced intersubtypic cross-protection: Mice immunized with A/California/7/04 (H3N2) + BCW were resistant to the infection by the highly pathogenic A/PR/8/34 (H1N1) virus.

Guinea pigs (Cavia porcellus) were treated with haloperidol (HP), and free radical (FR) and ferric reducing antioxidant power (FRAP) assays were used to determine oxidative stress levels. Furthermore, the superoxide dismutase (SOD), glutathione reductase (GR) and glutathione-S-transferase (GST) activity levels were detected and glucose levels and the reduced and oxidized glutathione (GSH/GSSG) ratio were measured in HP-treated and untreated guinea pigs. The present study demonstrated that the administration of HP causes significant oxidative stress in guinea pigs (P=0.022). In animals treated with HP, the activity of GST was significantly increased compared with a placebo (P= 0.007). The elevation of SOD and GR activity levels and increase in the levels of glutathione (GSH) in HP-treated animals were not statistically significant. In the HP-untreated animals, a significant positive correlation was observed between oxidative stress detected by the FR method and GST (r=0.88, P=0.008) and SOD (r=0.86, P= 0.01) activity levels, respectively. A significant negative correlation between the levels of plasma glucose and oxidative stress detected by the FRAP method was observed (r=-0.78, P=0.04). Notably, no significant correlations were observed in the treated animals. In the HP-treated group, two subgroups of animals were identified according to their responses to oxidative stress. The group with higher levels of plasma HP had higher enzyme activity and reactive oxygen species production compared with the group with lower plasma levels of HP. The greatest difference in activity (U/μl) between the two groups of animals was for GR.

Aim: The cardio-ankle vascular index (CAVI) is a novel non-invasive marker of arterial stiffness and atherosclerosis. The aim of this work was to examine whether the CAVI value in patients with dyslipidaemia (DLP) is increased by the presence of other cardiovascular risk factors: hypertension, diabetes mellitus, and smoking., Methods: A total of 392 subjects with DLP (166 male, 226 female), with a median age of 58.5 and 5-95 percentile range 32.2-73.9 years were examined. CAVI was measured using the VaSera 1500 system., Results: CAVI correlated significantly with age (p<0.001) and both systolic (p<0.001) and diastolic (p=0.002) blood pressure; higher values were found in men (p=0.034) than in women in the 56-65 age group. There was no significant difference in CAVI between smokers and non-smokers (p= 0.217) and between subjects with and without diabetes mellitus (p= 0.424). CAVI was significantly higher in subjects with hypertension than in the normotensive group (p<0.001) and in statin-treated subjects than in those without statins (p<0.001); however, CAVI values adjusted for age and sex did not differ significantly between these groups. Adjusted CAVI values were higher only in smokers than in non-smokers (former smokers) (p<0.001)., Conclusion: The study proves conclusively that the CAVI value in DLP patients is not significantly affected by hypertension and diabetes mellitus, but it is increased by smoking.

Muscarinic receptors (MR) are main cardioinhibitory receptors. We investigated the changes in gene expression, receptor number, echocardiography, muscarinic/adrenergic agonist/antagonist changes in heart rate (HR) and HR biorhythm in M(2) KO mice (mice lacking the main cardioinhibitory receptors) in the left ventricle (LV) and right ventricle (RV). We hypothesize that the disruption of M(2) MR, key players in parasympathetic bradycardia, would change the number of receptors with antagonistic effects on the heart (β(1)- and β(2)-adrenoceptors, BAR), while the function of the heart would be changed only marginally. We have found changes in LV, but not in RV: decrease in M(3) MR, β(1)- and β(2)-adrenoceptor gene expressions that were accompanied by a decrease in MR and BAR receptor binding. No changes were found both in LV systolic and diastolic function as assessed by echocardiography (e.g., similar LV end-systolic and end-diastolic diameter, fractional shortening, mitral flow characteristics, and maximal velocity in LV outflow tract). We have found only marginal changes in specific HR biorhythm parameters. The effects of isoprenaline and propranolol on HR were similar in WT and KO (but with lesser extent). Atropine was not able to increase HR in KO animals. Carbachol decreased the HR in WT but increased HR in KO, suggesting the presence of cardiostimulatory MR. Therefore, we can conclude that although the main cardioinhibitory receptors are not present in the heart, the function is not much affected. As possible mechanisms of almost normal cardiac function, the decreases of both β(1)- and β(2)-adrenoceptor gene expression and receptor binding should be considered.

Neuromuscular electrical stimulation (NMES) of leg muscles has been introduced in clinical practice as a rehabilitation (RHB) method in patients with chronic heart failure (CHF); however, the role of NMES on the reduction of arterial stiffness and autonomic disbalance in these patients has not yet been studied. Sixty-one patients with stable CHF (mean age 58.9 [2.1] years; mean ejection fraction 31 [4.2]%, New York Heart Association II-III) were randomly assigned into two groups. Patients in (i) exercise training group (ET; n = 30) underwent 12 weeks of bicycle ET (3 × 40 min/week); (ii) group NMES (n = 31) performed 12 weeks of NMES of quadriceps and calf muscles (frequency 10 Hz, mode "20 s on-20 s off," intensity 60 mA), 2 × 60 min/day. Noninvasive assessment of arterial stiffness was done using the cardio-ankle vascular index (CAVI). CAVI and heart rate variability (HRV) and ·VO(2peak) were evaluated before and after RHB program. Both types of RHB reduced significantly CAVI (ET from 9.6 [0.2] to 8.9 [0.2], P < 0.012; NMES from 9.3 [0.2] to 8.7 [0.2], P < 0.013), increased high frequency (HF) component of HRV (+65.6%; P = 0.001) and decreased ratio of low frequency (LF) component with HF component (LF/HF ratio) in group ET (-39.8%; P < 0.001). Changes of HRV parameters in group NMES were not significant; however, a marked tendency to autonomic stabilization was present. Both types of RHB led also to significant increase of ·VO(2peak) (ET from 18.7 [0.7] to 20.8 [0.7] mL/kg/min, P < 0.004; NMES from 17.3 [0.7] to 19.0 [0.7] mL/kg/min, P < 0.001). ET or NMES has been shown to improve significantly arterial stiffness and to stabilize autonomic balance., (© 2012, Copyright the Authors. Artificial Organs © 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

We investigated the role of beta3-adrenoceptors (AR) in cold stress (1 or 7 days in cold) in animals lacking main cardioinhibitive receptors-M2 muscarinic receptors (M(2)KO). There was no change in receptor number in the right ventricles. In the left ventricles, there was decrease in binding to all cardiostimulative receptors (beta1-, and beta2-AR) and increase in cardiodepressive receptors (beta3-AR) in unstressed KO in comparison to WT. The cold stress in WT animals resulted in decrease in binding to beta1- and beta2-AR (to 37%/35% after 1 day in cold and to 27%/28% after 7 days in cold) while beta3-AR were increased (to 216% of control) when 7 days cold was applied. MR were reduced to 46% and 58%, respectively. Gene expression of M2 MR in WT was not changed due to stress, while M3 was changed. The reaction of beta1- and beta2-AR (binding) to cold was similar in KO and WT animals, and beta3-AR in stressed KO animals did not change. Adenylyl cyclase activity was affected by beta3-agonist CL316243 in cold stressed WT animals but CL316243 had almost no effects on adenylyl cyclase activity in stressed KO. Nitric oxide activity (NOS) was not affected by BRL37344 (beta3-agonist) both in WT and KO animals. Similarly, the stress had no effects on NOS activity in WT animals and in KO animals. We conclude that the function of M2 MR is substituted by beta3-AR and that these effects are mediated via adenylyl cyclase rather than NOS.

In mammals, the circadian system is composed of the central clock in the hypothalamic suprachiasmatic nuclei and of peripheral clocks that are located in other neural structures and in cells of the peripheral tissues and organs. In adults, the system is hierarchically organized so that the central clock provides the other clocks in the body with information about the time of day. This information is needed for the adaptation of their functions to cyclically changing external conditions. During ontogenesis, the system undergoes substantial development and its sensitivity to external signals changes. Perinatally, maternal cues are responsible for setting the phase of the developing clock, while later postnatally, the LD cycle is dominant. The central clock attains its functional properties during a gradual and programmed process. Peripheral clocks begin to exhibit rhythmicity independent of each other at various developmental stages. During the early developmental stages, the peripheral clocks are set or driven by maternal feeding, but later the central clock becomes fully functional and begins to entrain the periphery. During the perinatal period, the central and peripheral clocks seem to be vulnerable to disturbances in external conditions. Further studies are needed to understand the processes of how the circadian system develops and what degree of plasticity and resilience it possesses during ontogenesis. These data may lead to an assessment of the contribution of disturbances of the circadian system during early ontogenesis to the occurrence of circadian diseases in adulthood., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aim: The cardio-ankle vascular index (CAVI) is a new non-invasive marker of arterial stiffness and atherosclerosis. The purpose of this study was to compare CAVI in patients with heterozygous familial hypercholesterolemia (FH) and in healthy controls., Methods: 82 FH subjects (27 males, 65 females), aged 53.7±13.6 years without clinical symptoms of cardiovascular diseases and 359 healthy controls (121 males, 238 females), aged 43.9±14.9 years, were examined. CAVI was measured using the system VaSera® 1500., Results: CAVI in FH patients was significantly higher (8.0±1.4) than in healthy subjects (7.5±1.3) p = 0.002; however, age, sex and BMI adjusted CAVI did not differ significantly (p = 0.061) between the FH group (7.5, CI: 7.3; 7.7) and control group (7.7, CI: 7.6; 7.7)., Conclusion: The study showed no significant difference in CAVI between heterozygous FH and healthy controls.

Hemodialyzed (HD) patients with end-stage renal disease (ESRD) exhibit lower fitness as a consequence of chronic uremic changes that trigger various structural, metabolic, and functional abnormalities in skeletal muscles. The aim of this randomized study was to compare the effect of rehabilitation (RHB) training on a bicycle ergometer and electromyostimulation (EMS) of leg extensors in HD patients with ESRD. Thirty-two HD patients (18 men/14 women; mean age 61.1 ± 8.8 years) were randomized into three groups: (i) exercise training (ET; n = 11) on bicycle ergometer 2 × 20 min; (ii) EMS (n = 11) where stimulation (10 Hz) of leg extensors was applied for 60 min; and (iii) controls (CON; n = 10) without exercise. Exercising was performed between the 2nd and the 3rd hour of HD, three times a week, 20 weeks in total. Ergometric test was performed in order to evaluate peak workload (W(peak)), 6-min corridor walking test (CWT) to evaluate the distance walked, and dynamometry of leg extensors to assess muscle power (F(max)). Urea clearance was monitored and expressed as standard parameters: spKt/V, spKt/V equilibrated (spKt/V-e), and the urea removal ratio (URR). Quality of life (QoL) was assessed by the questionnaire SF-36. A significant increase of F(max) (P = 0.040 in group ET; P = 0.032 in group EMS), of 6-min CWT (P < 0.001 in ET group; P = 0.042 in EMS group), and of W(peak) (P = 0.041 in ET group) was observed. In both exercising groups, significant increase of spKt/V, spKt/V-e, and URR was found as compared with initial values (P < 0.05). In both exercising groups, highly significant changes in summarized mental functions were found (P = 0.001); in summarized physical components, significant improvement was observed in the ET group (P = 0.006). Intradialytic RHB showed comparable positive effects on functional parameters, urea clearance, and QoL. Intradialytic EMS might represent wide therapeutic possibility in the near future., (© 2011, Copyright the Authors. Artificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

We have developed a generic platform to undertake the analysis of protein copy number from single cells. The approach described here is 'all-optical' whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by a shock wave caused by laser-induced microcavitation, and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. These results suggest that this is a viable method for measuring relative protein levels in single cells.

Extra-cellular adenosine is an important regulator of inflammatory responses. It is generated from released ATP by a cascade of ectoenzymes and degraded by adenosine deaminase (ADA). There are two types of enzymes with ADA activity: ADA1 and ADGF/ADA2. ADA2 activity originates from macrophages and dendritic cells and is associated with inflammatory responses in humans and rats. Drosophila possesses a family of six ADGF proteins with ADGF-A being the main regulator of extra-cellular adenosine during larval stages. Herein we present the generation of a GFP reporter for ADGF-A expression by a precise replacement of the ADGF-A coding sequence with GFP using homologous recombination. We show that the reporter is specifically expressed in aggregating hemocytes (Drosophila immune cells) forming melanotic capsules; a characteristic of inflammatory response. Our vital reporter thus confirms ADA expression in sites of inflammation in vivo and demonstrates that the requirement for ADA activity during inflammatory response is evolutionary conserved from insects to vertebrates. Our results also suggest that ADA activity is achieved specifically within sites of inflammation by an uncharacterized post-transcriptional regulation based mechanism. Utilizing various mutants that induce melanotic capsule formation and also a real immune challenge provided by parasitic wasps, we show that the acute expression of the ADGF-A protein is not driven by one specific signaling cascade but is rather associated with the behavior of immune cells during the general inflammatory response. Connecting the exclusive expression of ADGF-A within sites of inflammation, as presented here, with the release of energy stores when the ADGF-A activity is absent, suggests that extra-cellular adenosine may function as a signal for energy allocation during immune response and that ADGF-A/ADA2 expression in such sites of inflammation may regulate this role.

The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for β-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA , mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied., (© 2010 Landes Bioscience)

Thermoresponsive polymer micelles are promising drug and radionuclide carriers with a strong passive targeting effect into solid tumors. We have synthesized ABA triblock copolymers poly[2-methyl-2-oxazoline-block-(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazoline)-block-2-methyl-2-oxazoline]. These polymers are molecularly dissolved in aqueous millieu below the cloud point temperature (CPT) of the thermoresponsive central block and above CPT form polymer micelles at CMC 5-10 × 10(-5) g · mL(-1) with diameter ≈200 nm. The phenolic moiety introduced into the copolymer allowed radionuclide labeling with iodine-125 ongoing in good yield with sufficient in vitro stability under model conditions.

We have developed a radiolabeling strategy for synthetic polymers based on the formation of azo dye usable for both covalent and chelating labeling modalities under mild conditions. Poly[N-(2-hydroxypropyl)methacrylamide] and poly(N-isopropyl acrylamide) were used as model polymers. N-methacryloyl tyrosinamide was introduced into the polymers and the phenolic moiety was then reacted with diazotized chelator precursors. The conjugates were radiolabeled with both the covalently bound (iodine-125) and chelated (indium-111) radionuclides in high yields and sufficient in vitro stability of the labels was proven., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

We tested the hypothesis that single and repeated immobilization stress affect densities of alpha(1)-adrenoceptor (alpha(1)-AR) and beta-AR subtypes, muscarinic receptors (MR), adenylyl cyclase activity (AC) and phospholipase C activity (PLC) in lungs of male and female wild type (WT) and corticotropin-releasing hormone gene (CRH-knockout (KO)) disrupted mice. We found sex differences in the basal levels of alpha(1)-AR subtypes (females had 2-3 times higher density of receptors than males) and MR (males had twice the density found in females). In marked contrast, beta-AR subtype densities did not differ between sexes. CRH gene disruption decreased all three studied receptors in intact mice (to 20-50% of WT) in both sexes (except beta(1)-AR in females). Stress induced sexually dimorphic responses, while all alpha(1)-AR subtypes decreased in females (to 30% of control approximately), only alpha(1A)-AR level diminished (about 50%) in males. beta(1)-AR decreased in males (to about 40%) but remained stable in females. beta(2)-AR diminished in females (to about 20-60%) and also in males (to about 30-60%). MR decreased in both sexes (approximately to 50%). AC activity diminished in males (to < 50%) while PLC activity was not changed. In CRH-KO mice, the stress response was severely diminished. Paradoxically, the receptor response to stress was less affected by CRH-KO in males than in females. AC activity did not change in CRH-KO mice. In conclusion, in mice the stress reaction is sexually dimorphic and an intact hypothalamo-pituitary-adrenocortical system is required for the normal reaction of pulmonary adrenergic and MR to stress.

In order to evaluate the potential risks of human granulocytic anaplasmosis in Slovakia, blood and serum samples of hunters and foresters from the northern parth of Slovakia were tested. We present the first case of HGA from Slovakia confirmed by nested PCR amplification of the 16S rRNA gene fragment of A. phagocytophilum.