Setting the biological time in central and peripheral clocks during ontogenesis

In mammals, the principal circadian clock within the suprachiasmatic nucleus (SCN) entrains the phase of clocks in numerous peripheral tissues and controls the rhythmicity in various body functions. During ontogenesis, the molecular mechanism responsible for generating circadian rhythmicity develops gradually from the prenatal to the postnatal period. In the beginning, the maternal signals set the phase of the newly developing fetal and early postnatal clocks, whereas the external light-dark cycle starts to entrain the clocks only later. This minireview discusses the complexity of signaling pathways from mothers and the outside world to the fetal and newborn animals' circadian clocks.     

Acinetobacter calcoaceticus–baumannii Complex Strains Induce Caspase-Dependent and Caspase-Independent Death of Human Epithelial Cells

We investigated interactions of human isolates of Acinetobacter calcoaceticus-baumannii complex strains with epithelial cells. The results showed that bacterial contact with the cells as well as adhesion and invasion were required for induction of cytotoxicity. The infected cells revealed hallmarks of apoptosis characterized by cell shrinking, condensed chromatin, and internucleosomal fragmentation of nuclear DNA. The highest apoptotic index was observed for 4 of 10 A. calcoaceticus and 4 of 7 A. baumannii strains. Moreover, we observed oncotic changes: cellular swelling and blebbing, noncondensed chromatin, and the absence of DNA fragmentation. The highest oncotic index was observed in cells infected with 6 A. calcoaceticus isolates. Cell-contact cytotoxicity and cell death were not inhibited by the pan-caspase inhibitor z-VAD-fmk. Induction of oncosis was correlated with increased invasive ability of the strains. We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death. Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨ(m)). Bacterial infection caused generation of nitric oxide and reactive oxygen species. This study indicated that Acinetobacter spp. induced strain-dependent distinct types of epithelial cell death that may contribute to the pathogenesis of bacterial infection.     

Externalization of phosphatidylserine from inner to outer layer may alter the effect of plant sterols on human erythrocyte membrane - The Langmuir monolayer studies

One of the factors, which can strongly modify the cell membrane composition, is disordering in membrane asymmetry, resulting from redistribution of lipids from inner to outer layer. Such a disturbance may affect the behavior of various biologically active compounds incorporating into membranes. In this contribution, the relationship between the amounts of phosphatidylserine (PS) in the model outer layer of human erythrocyte (RBC) membrane and the effect induced by a plant sterol (β-sitosterol) was verified. The experiments were performed on multicomponent Langmuir films imitating red blood cell (RBC) membrane, differing in the contents of PS (0%; 5% and 10%) into which the plant sterol was incorporated in various concentrations. The analysis of experimental results (surface pressure-area isotherms complemented with Brewster Angle Microscopy (BAM) proved that the presence of phosphatidylserine molecules, depending on their contents in the mixed monolayer mimicking RBC membrane, changes its properties and exerts influence on the effect of plant sterol on the model system. The addition of phytosterol into the monolayer that lacks or contains only 5% of PS was found to be of rather weak effect on the properties of the system. However, in the case of the model membrane containing the increased amount (10%) of PS, the incorporation of plant sterol strongly affects the interactions between molecules and caused thermodynamic destabilization of the monolayer imitating RBC membrane. These results allow one to suggest that externalization of phosphatidyserine to the outer membrane leaflet may differentiate the effect of plant sterols on cell membranes of various origins.     

PPAR-alpha activation as a preconditioning-like intervention in rats in vivo confers myocardial protection against acute ischaemia-reperfusion injury: involvement of PI3K-Akt

Peroxisome proliferator-activated receptors (PPAR) regulate the expression of genes involved in lipid metabolism, energy production, and inflammation. Their role in ischaemia-reperfusion (I/R) is less clear, although research indicates involvement of PPARs in some forms of preconditioning. This study aimed to explore the effects of PPAR-α activation on the I/R injury and potential cardioprotective downstream mechanisms involved. Langendorff-perfused hearts of rats pretreated with the selective PPAR-α agonist WY-14643 (WY, pirinixic acid; 3 mg·(kg body mass)·day(-1); 5 days) were subjected to 30 min ischaemia - 2 h reperfusion with or without the phosphatidylinositol 3-kinase (PI3K)-Akt inhibitor wortmannin for the evaluation of functional (left ventricular developed pressure, LVDP) recovery, infarct size (IS), and reperfusion-induced arrhythmias. A 2-fold increase in baseline PPAR-α mRNA levels (qPCR) in the WY-treated group and higher post-I/R PPAR-α levels compared with those in untreated controls were accompanied by similar changes in the expression of PPAR-α target genes PDK4 and mCPT-1, regulating glucose and fatty acid metabolism, and by enhanced Akt phosphorylation. Post-ischaemic LVDP restoration in WY-treated hearts reached 60% ± 9% of the pre-ischaemic values compared with 24% ± 3% in the control hearts (P < 0.05), coupled with reduced IS and incidence of ventricular fibrillation that was blunted by wortmannin. Results indicate that PPAR-α up-regulation may confer preconditioning-like protection via metabolic effects. Downstream mechanisms of PPAR-α-mediated cardioprotection may involve PI3K-Akt activation.     

Ephedrine accelerates psychomotor recovery from anesthesia in macaque monkeys

Background Ephedrine is used in treatment of hypotension during anesthesia. We investigated its effects on the psychomotor recovery and its potential adverse reactions on cardiorespiratory functions in rhesus monkeys. Methods The monkeys received 50 μg/kg medetomidine, 2.0 mg/kg S‐ketamine with 150 IU hyaluronidase i.m. Pulse rate, blood pressure and saturation of haemoglobin were monitored for 20 minutes. Thereafter, 1 mg/kg of ephedrine or a placebo was administered i.m. and behavioural changes, pulse rate, blood pressure and saturation of haemoglobin were monitored every 5 minutes. Results Ephedrine shortened recovery from anaesthesia from 80.4 ± 25.8 to 14.83 ± 13.70 minutes. Ephedrine also increased oxygen saturation of haemoglobin and systolic blood pressure and caused significant decrease in pulse rate 5 minutes after its administration. Conclusions Ephedrine can be successfully used to accelerate psychomotor recovery after the use of common anesthetic protocols combining dissociative anesthetic agent and alpha 2‐adrenoceptor agonist in primates.     

Central carbon metabolism influences fidelity of DNA replication in Escherichia coli

Recent studies indicated that there is a direct link between central carbon metabolism (CCM) and initiation and elongation of DNA replication in Eschericha coli. Namely, effects of certain mutations in genes coding for replication proteins (dnaA, dnaB, dnaE, dnaG, and dnaN) could be specifically suppressed by deletions of some genes, whose products are involved in CCM reactions (pta, ackA, pgi, tktB, and gpmA). Here, we demonstrate that the link between CCM and DNA synthesis can be extended to the DNA replication fidelity, as we report changes of the mutator phenotypes of E. coli dnaQ49 and dnaX36 mutants (either suppression or enhancement) by dysfunctions of zwf, pta, ackA, acnB, and icdA genes. Overexpression of appropriate wild-type CCM genes in double mutants resulted in reversions to the initial mutator phenotypes, indicating that the effects were specific. Moreover, the observed suppression and enhancement effects were not caused by changes in bacterial growth rates. These results suggest that there is a genetic correlation between CCM and DNA replication fidelity in E. coli, apart from the already documented link between CCM and DNA replication initiation control and elongation efficiency.     

Microbotryum heliospermae, a new anther smut fungus parasitic on Heliosperma pusillum in the mountains of the European Alpine System

The members of the smut genus Microbotryum are pathogens of a wide range of host plant species from nine dicotyledonous families. Within the genus, the species sporulating in anthers of Caryophyllaceae form a monophyletic group that in recent years attracted much interest in various biological studies. The phylogenetic framework developed for species delimitation within Microbotryum revealed that high level host-specificity is a major feature of most caryophyllaceous anther smuts. However, the great number of anther smut specimens on diverse host plant species reported worldwide has still not been included in phylogenetic analyses due to the inaccessibility of recently collected specimens, and thus many species remain still undiscovered. In this study, anther smut specimens on Heliosperma pusillum originating from all main mountain ranges of the European Alpine System were examined using partial rDNA sequence and/or morphological analyses. The investigation revealed that all specimens are morphologically uniform and phylogenetically represent a monophyletic lineage, sister to Microbotryum lagerheimii complex on Atocion rupestre/Silene lacera/Silene vulgaris/Viscaria vulgaris. This lineage cannot be attributed to any of the previously described species, and therefore the smut in anthers of H. pusillum is described and illustrated here as a new species, Microbotryum heliospermae. The species is known from subalpine zone of the Alps, the Carpathians, the Dinaric Alps, and the Pyrenees, inhabiting host plants growing in open spring communities or semihumid mountain meadows.     

Long-term follow-up in adult patients with low-grade glioma (WHO II) postoperatively irradiated. Analysis of prognostic factors

AimTo report the long-term follow-up of a cohort of adult patients with LGG post-operatively irradiated in one institution, and to identify prognostic factors for progression free survival.BackgroundThere is little consensus about the optimal treatment for low-grade glioma (LGG), and the clinical management of LGG is one of the most controversial areas in neurooncology. Radiation therapy is one option for treatment of patients with LGG, whereas other options include postoperative observation.Materials and methodsBetween 1975 and 2005, 180 patients with LGG (WHO II) received postoperative irradiation after non radical (subtotal or partial) excision. Patients had to be 18 years of age or older, and have histologic proof of supratentorial fibrillary (FA), protoplasmic (PA) or gemistocytic astrocytoma (GA). Radiotherapy was given within 3–10 weeks after surgery. Treatment fields were localized and included the preoperative tumor volume, with a 1–2 cm margin, treated to a total dose of 50–60 Gy in 25–30 fractions over 5–6 weeks.ResultsActuarial ten-year progression free survival (APFS) in the whole group was 19%. The worse prognosis was observed in patients with GA. Ten-year APFS rates for GA, PA and FA were 10%, 18% and 22%, respectively.ConclusionThe findings from our long-term cohort of 180 patients with LGG confirmed by uni- and multivariate analysis demonstrated that only astrocytoma histology significantly determined the prognosis. The best survival was observed in patients with the fibrillary variant, and the worst for the gemistocytic one.     

Antibiotic transport in resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic accumulation with single cell resolution

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack.