The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii

Legionella pneumophila and Coxiella burnetii have been shown to utilize the icm/dot type IV secretion system for pathogenesis and recently a large number of icm/dot-translocated substrates were identified in L. pneumophila. Bioinformatic analysis has revealed that 13 of the genes encoding for L. pneumophila-translocated substrates and five of the C. burnetii icm/dot genes, contain a conserved regulatory element that resembles the target sequence of the PmrA response regulator. Experimental analysis which included the construction of a L. pneumophila pmrA deletion mutant, intracellular growth analysis, comparison of gene expression between L. pneumophila wild type and the pmrA mutant, construction of mutations in the PmrA conserved regulatory element, controlled expression studies as well as mobility shift assays, demonstrated the direct relation between the PmrA regulator and the expression of L. pneumophila icm/dot-translocated substrates and several C. burnetii icm/dot genes. Furthermore, genomic analysis identified 35 L. pneumophila and 68 C. burnetii unique genes that contain the PmrA regulatory element and few of these genes from L. pneumophila were found to be new icm/dot-translocated substrates. Our results establish the PmrA regulator as a fundamental regulator of the icm/dot type IV secretion system in these two bacteria.     

Assessing the agronomic and environmental effects of the application of cattle manure compost on soil by multivariate methods

Multivariate analysis was used for interpreting data from a pot experiment using samples of three Spanish soils. Samples of soil fertilized with compost were compared with untreated control samples. We also compared the effect of adding the compost to soil with a controlled moisture content of 50% of its water holding capacity (WHC), and to a near-saturated soil (95% WHC). Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were used; they perfectly differentiated sample groups both as a function of the treatment applied and by sampling date. The compost samples were characterized by higher pH, electrical conductivity (EC), organic matter (OM) content and cation exchange capacity (CEC), together with nutrient concentrations than the control pots. The pots with a soil–compost mixture at 95% WHC presented lower values of EC, CEC, inorganic N, K, Na and B than the mixtures at 50% WHC. Multivariate methods may therefore be useful for the analysis and interpretation of a large number of data in soil research.     

Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.     

Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 approximately 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.     

An early-flowering genotype of<Emphasis Type="Italic">Populus</Emphasis>

Unlike herbaceous, annual crops, trees are not highly domesticated and, therefore, have wild relatives with which they are interfertile. They are also long-lived perennials that produce copious amounts of pollen and seed, which are often disseminated over considerable distances by the wind. Federal regulators have made it clear that before transgenic trees can be grown commercially in the U.S., it will be necessary to develop a strategy to mitigate the risk of transgene spread into the environment. One way to satisfy this requirement is to genetically engineer reproductive sterility. Because of its many useful attributes, poplar has becomethe model tree species for research community. However, because of its relatively long juvenile period, the development of a reliable sterility system for poplar is taking longer than expected. By having an early-flowering genotype of poplar, it will be possible to make much faster progress in our efforts to develop a reliable transgene-confinement system. We have identified a genotype ofPopulus alba that can be induced to flower within nine months of being regenerated.     

Phylogeography of Y-chromosome haplogroup I reveals distinct domains of prehistoric gene flow in europe

To investigate which aspects of contemporary human Y-chromosome variation in Europe are characteristic of primary colonization, late-glacial expansions from refuge areas, Neolithic dispersals, or more recent events of gene flow, we have analyzed, in detail, haplogroup I (Hg I), the only major clade of the Y phylogeny that is widespread over Europe but virtually absent elsewhere. The analysis of 1,104 Hg I Y chromosomes, which were identified in the survey of 7,574 males from 60 population samples, revealed several subclades with distinct geographic distributions. Subclade I1a accounts for most of Hg I in Scandinavia, with a rapidly decreasing frequency toward both the East European Plain and the Atlantic fringe, but microsatellite diversity reveals that France could be the source region of the early spread of both I1a and the less common I1c. Also, I1b*, which extends from the eastern Adriatic to eastern Europe and declines noticeably toward the southern Balkans and abruptly toward the periphery of northern Italy, probably diffused after the Last Glacial Maximum from a homeland in eastern Europe or the Balkans. In contrast, I1b2 most likely arose in southern France/Iberia. Similarly to the other subclades, it underwent a postglacial expansion and marked the human colonization of Sardinia ~9,000 years ago.     

Modulation of adenyl cyclase activity in the gills of Tapes philippinarum

Adenyl cyclase (AC) plays a pivotal role in cell signaling. The AC system of bivalves has received little attention so far, and our study has been addressed to the characterization of AC properties in the gills of T. philippinarum. The enzyme showed a Km value of 0.77 mM for ATP in the presence of 5 mM Mg2+; in the absence of agonists, it was poorly affected by GTP, while it was stimulated by GTPgammaS and GppNHp up to 14-fold and 4-fold, respectively. Similarly to other invertebrates, the enzyme activity was scarcely stimulated by forskolin. The receptor agonist serotonin (5-HT) significantly stimulated the AC activity, and the pharmacological profile of the 5-HT receptor/s was as follows: (+)butaclamol > dihydroergocryptine > methysergide > prazosin > yohimbine. The AC activity was assessed in vitro in the presence of tributyltin chloride and HgCl2, which reduced the AC activity only at the highest dose tested (10-100 microM). Our data indicate the presence of a membrane-bound AC in gill membranes of T. philippinarum, coupled to Gs proteins and to a specific class of 5-HT receptors. Such receptors show a pharmacological profile slightly different from that reported for 5-HT invertebrate receptors cloned so far.     

Cost-Utility Analysis of Lopinavir/Ritonavir versus Atazanavir + Ritonavir Administered as First-Line Therapy for the Treatment of HIV Infection in Italy: From Randomised Trial to Real World

Objective

To estimate the lifetime cost utility of two antiretroviral regimens (once-daily atazanavir plus ritonavir [ATV+r] versus twice-daily lopinavir/ritonavir [LPV/r]) in Italian human immunodeficiency virus (HIV)-infected patients naïve to treatment.

Design

With this observational retrospective study we collected the clinical data of a cohort of HIV-infected patients receiving first-line treatment with LPV/r or ATV+r.

Methodology

A Markov microsimulation model including direct costs and health outcomes of first- and second-line highly active retroviral therapy was developed from a third-party (Italian National Healthcare Service) payer’s perspective. Health and monetary outcomes associated with the long-term use of ATV+r and LPV/r regimens were evaluated on the basis of eight health states, incidence of diarrhoea and hyperbilirubinemia, AIDS events, opportunistic infections, coronary heart disease events and, for the first time in an economic evaluation, chronic kidney disease (CKD) events. In order to account for possible deviations between real-life data and randomised controlled trial results, a second control arm (ATV+r 2) was created with differential transition probabilities taken from the literature.

Results

The average survival was 24.061 years for patients receiving LPV/r, 24.081 and 24.084 for those receiving ATV+r 1 and 2 respectively. The mean quality-adjusted life-years (QALYs) were higher for the patients receiving LPV/r than those receiving ATV+r (13.322 vs. 13.060 and 13.261 for ATV+r 1 and 2). The cost-utility values were 15,310.56 for LPV/r, 15,902.99 and 15,524.85 for ATV+r 1 and 2.

Conclusions

Using real-life data, the model produced significantly different results compared with other studies. With the innovative addition of an evaluation of CKD events, the model showed a cost-utility value advantage for twice-daily LPV/r over once-daily ATV+r, thus providing evidence for its continued use in the treatment of HIV.