Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90.6% of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.     

Rate of increase in circulating IL-7 and loss of IL-7Ralpha expression differ in HIV-1 and HIV-2 infections: two lymphopenic diseases with similar hyperimmune activation but distinct outcomes

IL-7 is a nonredundant cytokine for T cell homeostasis. Circulating IL-7 levels increase in lymphopenic clinical settings, including HIV-1 infection. HIV-2 infection is considered a "natural" model of attenuated HIV disease given its much slower rate of CD4 decline than HIV-1 and limited impact on the survival of the majority of infected adults. We compared untreated HIV-1- and HIV-2-infected patients and found that the HIV-2 cohort demonstrated a delayed increase in IL-7 levels during the progressive depletion of circulating CD4 T cells as well as a dissociation between the acquisition of markers of T cell effector differentiation and the loss of IL-7Ralpha expression. This comparison of two persistent infections associated with progressive CD4 depletion and immune activation demonstrates that a better prognosis is not necessarily associated with higher levels of IL-7. Moreover, the delayed increase in IL-7 coupled with sustained expression of IL-7Ralpha suggests a maximization of available resources in HIV-2. The observation that increased IL-7 levels early in HIV-1 infection were unable to reduce the rate of CD4 loss and the impaired expression of the IL-7Ralpha irrespective of the state of cell differentiation raises concerns regarding the use of IL-7 therapy in HIV-1 infection.     

Street heroin induces mitochondrial dysfunction and apoptosis in rat cortical neurons

Cortical function has been suggested to be highly compromised by repeated heroin self-administration. We have previously shown that street heroin induces apoptosis in neuronal-like PC12 cells. Thus, we analysed the apoptotic pathways involved in street heroin neurotoxicity using primary cultures of rat cortical neurons. Our street heroin sample was shown to be mainly composed by heroin, 6-monoacetylmorphine and morphine. Exposure of cortical neurons to street heroin induced a slight decrease in metabolic viability, without loss of neuronal integrity. Early activation of caspases involved in the mitochondrial apoptotic pathway was observed, culminating in caspase 3 activation, Poly-ADP Ribose Polymerase (PARP) cleavage and DNA fragmentation. Apoptotic morphology was completely prevented by the non-selective caspase inhibitor z-VAD-fmk, indicating an important role for caspases in neurodegeneration induced by street heroin. Ionotropic glutamate receptors, opioid receptors and oxidative stress were not involved in caspase 3 activation. Interestingly, street heroin cytotoxicity was shown to be independent of a functional mitochondrial respiratory chain, as determined using NT-2 rho(0) cells. Nonetheless, in street heroin-treated cortical neurons, cytochrome c was released, accompanied by a decrease in mitochondrial potential and Bcl-2/Bax. Pure heroin hydrochloride similarly decreased metabolic viability but only slightly activated caspase 3. Altogether, our data suggest an important role for mitochondria in mediating street heroin neurotoxic effects.     

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.     

Life cycle assessment of a multi-material car component

Background, Aims and Scope In recent years, the automotive industry has been experiencing an increasing concern with environmental requirements. A particular focus is being given to light-weighting of cars, to reducing fuel consumption and to the use of different recycling materials. Consequently, decisions on product design and development must involve economic and technological as well as environmental considerations. In adequate conditions, the LCA methodology enables one to assist an effective integration of the environmental considerations in the decision-making process [1]. In this paper, a multi-material car component which is part of the current automotive brake system, has been modified by its original manufacturer. Such a modification included the use of a new multi-material injection moulding process and the consumption of recyclable materials. The new and the current component were comparatively assessed throughout their life cycles in order to evaluate their respective environmental impacts and, thus, to verify if the new component offers a lower environmental load. The results described in this paper are part of the outcome of a broader research project involving industrial companies, university, technological centres and research institutes based in Portugal, Spain and Germany. Main Features The car component under focus has four subcomponents whose base materials consist of steel and plastic. The LCA methodology is used to evaluate two scenarios describing the new car component, on the one hand, and the reference scenario, which consists of the existing car component, on the other. The former results from the selection of new subcomponents materials, aiming to use a new production process together with a recycling strategy. Results and Discussion The inventory analysis shows a lower energy consumption in the alternative scenario (4.2 MJ) compared to the reference scenario (6.1 MJ). Most of that energy is still non-renewable, relating in particular to crude consumption in the car use phase and in the production phase (transports and plastics production). The life cycle inventory analysis indicates also that the alternative scenario has lower air emissions of CO₂, CO, NO_x, SO_x, NM VOC and PM10, as well as lower solid wastes and water emissions of oils and BOD5. Otherwise, the water emissions of undissolved substances and COD are higher for the alternative scenario. Most of the energy consumed and the air pollutants inventoried occur as a consequence of the use phase. Otherwise, for most of the life cycle water emissions inventoried and solid wastes, the production phase is the major contributor. The impact assessment, performed with the CML method, allows one to conclude that the alternative scenario exhibits lower results in all the impact categories. Both scenarios have similar environmental profiles, being: (i) the use phase, the major contributor for the abiotic depletion, global warming, photochemical oxidation, acidification and eutrophication; and (ii) the production phase, the main contributor for ozone depletion, human toxicity, fresh water aquatic ecotoxicity, marine aquatic ecotoxicity and terrestrial ecotoxicity. The sensitivity analysis, with respect to the fuel consumption reduction value, the impact assessment method and the final disposal scenario, performed in this study allows one to confirm, as a main conclusion, that the alternative scenario is environmentally preferable to the reference scenario. Conclusion The results obtained through the application of the LCA methodology enable one to conclude that the alternative component has a lower environmental load than the reference component. Recommendations and Perspectives Considering that the time required for the inventory data collection is a critical issue in LCA practise, the insights provided by this particular case study are likely to be useful to product developers in the car component manufacturing industry, particularly to brake system manufacturers supporting the environmental design within the sector.     

Enzymatic treatments of pulp using laccase and hydrophobic compounds

The aim of this work was to develop an innovative method for the internal sizing of paper by use of laccase and hydrophobic compounds. Nine different products containing hydrophobic moieties were tested in combination with laccase derived from Trametes villosa on Eucalyptus globulus kraft pulp in order to assess their internal sizing capability. The strongest internal sizing effect was obtained with lauryl gallate (LG). Heat treatment of the handsheets was found to increase the resistance to water absorption of internally sized samples significantly. Tests were conducted under variable operating conditions, including enzyme and reactant doses and treatment time. In addition to altering the water absorption rate, internal sizing with the laccase–LG treatments was found to affect the mechanical and optical properties of the handsheets. As shown in this work, treatments based on laccase and a hydrophobic compound (particularly lauryl gallate), can provide a new, effective biotechnological method for the internal sizing of paper.     

Fertilization and allelopathy modify Pinus halepensis saplings crown acclimation to shade

Pinus halepensis Mill., is a Mediterranean pioneer forest species with shade-intolerant features. The purpose of this study is to better understand how stand fertility and allelopathic properties of adult trees influence shade acclimation of saplings. Crown growth and morphological plasticity were studied under different light, fertilization, and allelopathic conditions in a nursery experiment. We tested whether shade-acclimation capacity increases with fertilization, and is affected by autotoxicity due to pine leachates. We examined stem diameter, and crown characteristics (length, width, shape, and density) in a factorial experiment with two levels for each tested factor: light (full and 20% reduced light), fertilization (low and high rate of NPK fertilizer) and allelopathy (control and allelopathic leachates uptake). In our study, shading induced a significantly higher crown length, width, and surface. Fertilization strongly increased crown length and vertical expended crown shape (the ratio crown length/crown width). Leachates uptake reduced crown length and density, highlighting an autotoxicity phenomenon. We concluded that P. halepensis saplings presented a shade-avoiding syndrome and that the crown shade-acclimation response increased with fertilization but was severely compromised by autotoxicity. We finally discuss the role of fertilization and allelopathy in early P. halepensis acclimation ability.     

Throwing velocities, anthropometric characteristics, and efficacy indices of women's European water polo subchampions

Water polo is a team sport characterized by a high-intensity, intermittent activity, where technical and tactical aspect are of a great importance. For that reason, the main aim of this study was to define the anthropometrical characteristics, maximum isometric grip strength, training and competition throwing velocities, and the efficacy indices in female high-level water polo players. A second purpose was to examine the differences between the throwing velocities in training vs. European championships in the water polo female national team. Ten elite trained female water polo players participated in this study. Before the competitive phase of their season, the following measures were taken: standard anthropometry, static and dynamic training throwing velocities, and hand-grip dynamometry. In the competitive phase, efficacy indices, average and maximum throwing velocities from all the participants were also determined. Significant differences (p ≤ 0.05) were found between different training situations and different competitive throwing velocities. We concluded that elite female water polo players modify their throwing velocity depending if the throw is performed during training or competitive situation.     

The adiponectin receptor homologs in C. elegans promote energy utilization and homeostasis

Adiponectin is an adipokine with insulin-sensitising actions in vertebrates. Its receptors, AdipoR1 and AdipoR2, are PAQR-type proteins with 7-transmembrane domains and topologies reversed that of GPCR's, i.e. their C-termini are extracellular. We identified three adiponectin receptor homologs in the nematode C. elegans, named paqr-1, paqr-2 and paqr-3. These are differently expressed in the intestine (the main fat-storing tissue), hypodermis, muscles, neurons and secretory tissues, from which they could exert systemic effects. Analysis of mutants revealed that paqr-1 and -2 are novel metabolic regulators in C. elegans and that they act redundantly but independently from paqr-3. paqr-2 is the most important of the three paqr genes: mutants grow poorly, fail to adapt to growth at low temperature, and have a very high fat content with an abnormal enrichment in long (C20) poly-unsaturated fatty acids when combined with the paqr-1 mutation. paqr-2 mutants are also synthetic lethal with mutations in nhr-49, sbp-1 and fat-6, which are C. elegans homologs of nuclear hormone receptors, SREBP and FAT-6 (a Δ9 desaturase), respectively. Like paqr-2, paqr-1 is also synthetic lethal with sbp-1. Mutations in aak-2, the C. elegans homolog of AMPK, or nhr-80, another nuclear hormone receptor gene, suppress the growth phenotype of paqr-2 mutants, probably because they restore the balance between energy expenditure and storage. We conclude that paqr-1 and paqr-2 are receptors that regulate fatty acid metabolism and cold adaptation in C. elegans, that their main function is to promote energy utilization rather than storage, and that PAQR class proteins have regulated metabolism in metazoans for at least 700 million years.