Induction of apoptosis by mercury compounds depends on maturation and is not associated with microglial activation

The earliest sign of neurotoxicity observed after exposure of three-dimensional brain cell cultures to low concentrations of mercury compounds is a microglial reaction. We hypothesized that an induction of apoptosis by mercury compounds could be an activating signal of the microglial reaction. Aggregating brain cell cultures of fetal rat telencephalon were treated for 10 days with either mercury chloride or monomethylmercury chloride at noncytotoxic concentrations during two developmental periods: from day 5 to 15, corresponding to an immature stage, and from day 25 to 35 corresponding to a mature stage. Apoptosis was evaluated by the TUNEL technique. It was found that both mercury compounds caused a significant increase in the number of apoptotic cells, but exclusively in immature cultures exhibiting also spontaneous apoptosis. Double staining by the TUNEL technique combined with either neuronal or astroglial markers revealed that the proportion of cells undergoing apoptosis was highest for astrocytes. Furthermore neither an association nor a colocalization was found between apoptotic cells and microglial cells. In conclusion, it appears that the induction of apoptosis by mercury compounds in immature cells is only an acceleration of a spontaneously occurring process, and that it is not a directly related to the early microglial reaction.

Combining blockers of the renin-angiotensin system or increasing the dose of an angiotensin II receptor antagonist in proteinuric patients: a randomized triple-crossover study.

Objective The goal of this study was to investigate whether increasing the dose of an angiotensin II receptor blocker (ARB) provides as much benefits as combining the ARB with an angiotensin-converting enzyme inhibitor (ACEI) in terms of blood pressure (BP) control and urinary albumin excretion (UAE) in hypertensive patients with a proteinuria.Methods We enrolled 20 hypertensive patients with proteinuric nephropathies and a reduced renal function in a randomized, 12-month, triple-crossover, prospective, open-label study to compare the effects of a regular dose of losartan (Los 100mg q.d., LOS100) vs. a high dose of losartan (Los 100mg b.i.d., LOS200) vs. losartan 100mg q.d. associated with lisinopril 20 mg q.d. (LOS100 + LIS20). Each treatment was given for 8 weeks with a 4-week initial run-in period and 2 weeks of washout between each treatment phases. 24 h UAE and ambulatory BP were measured during the running phase and at the end of each treatment period.Results Compared to pretreatment, 24 h SBP and DBP were reduced by 10/5 +/- 7/4 mmHg with LOS100 (P=0.023 vs. baseline) and, respectively, 13/6 +/- 12/5 mmHg with LOS200 (P=0.011) and 19/9 +/- 15/8 mmHg with LOS100+LIS20 (P < 0.01). UAE decreased significantly with LOS100 and to an even greater degree with LOS200 and LOS100+LIS20 (P < 0.01 vs. baseline for both and P=0.032, LOS100+LIS20 vs. LOS200). The combination had a greater impact in patients with a high baseline proteinuria as suggested by a nonparallel leftward shift of the relationship between the changes in UAE induced by the combination and those induced by LOS200. The high dose of losartan was better tolerated than the combination.Conclusion Increasing the dose of losartan from 100mg once daily to 100mg twice a day enables to obtain a greater decrease in BP and proteinuria and is better tolerated than combining the ARB with lisinopril, though the high dose appears to be slightly less effective than the combination in patients with a marked proteinuria. J Hypertens 29: 1228-1235 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Rivastigmine for HIV-associated neurocognitive disorders: A randomized crossover pilot study.

OBJECTIVE: To assess the efficacy and safety of rivastigmine for the treatment of HIV-associated neurocognitive disorders (HAND) in a cohort of long-lasting aviremic HIV+ patients. METHODS: Seventeen aviremic HIV+ patients with HAND were enrolled in a randomized, double-blind, placebo-controlled, crossover study to receive either oral rivastigmine (up to 12 mg/day for 20 weeks) followed by placebo (20 weeks) or placebo followed by rivastigmine. Efficacy endpoints were improvement on rivastigmine in the Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-Cog) and individual neuropsychological scores of information processing speed, attention/working memory, executive functioning, and motor skills. Measures of safety included frequency and nature of adverse events and abnormalities on laboratory tests and on plasma concentrations of antiretroviral drugs. Analyses of variance with repeated measures were computed to look for treatment effects. RESULTS: There was no change on the primary outcome ADAS-Cog on drug. For secondary outcomes, processing speed improved on rivastigmine (Trail Making Test A: F(1,13) = 5.57, p = 0.03). One measure of executive functioning just failed to reach significance (CANTAB Spatial Working Memory [strategy]: F(1,13) = 3.94, p = 0.069). No other change was observed. Adverse events were frequent, but not different from those observed in other populations treated with rivastigmine. No safety issues were recorded. CONCLUSIONS: Rivastigmine in aviremic HIV+ patients with HAND seemed to improve psychomotor speed. A larger trial with the better tolerated transdermal form of rivastigmine is warranted. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that rivastigmine is ineffective for improving ADAS-Cog scores, but is effective in improving some secondary outcome measures in aviremic HIV+ patients with HAND.

Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon.

A three-dimensional cell culture system was used as a model to study the influence of low levels of mercury in the developing brain. Aggregating cell cultures of fetal rat telencephalon were treated for 10 days either during an early developmental period (i.e., between days 5 and 15 in vitro) or during a phase of advanced maturation (i.e., between days 25 and 35) with mercury. An inorganic (HgCl2) and an organic mercury compound (monomethylmercury chloride, MeHgCl) were examined. By monitoring changes in cell type-specific enzymes activities, the concentration-dependent toxicity of the compounds was determined. In immature cultures, a general cytotoxicity was observed at 10(-6) M for both mercury compounds. In these cultures, HgCl2 appeared somewhat more toxic than MeHgCl. However, no appreciable demethylation of MeHgCl could be detected, indicating similar toxic potencies for both mercury compounds. In highly differentiated cultures, by contrast, MeHgCl exhibited a higher toxic potency than HgCl2. In addition, at 10(-6) M, MeHgCl showed pronounced neuron-specific toxicity. Below the cytotoxic concentrations, distinct glia-specific reactions could be observed with both mercury compounds. An increase in the immunoreactivity for glial fibrillary acidic protein, typical for gliosis, could be observed at concentrations between 10(-9) M and 10(-7) M in immature cultures, and between 10(-8) M and 3 x 10(-5) M in highly differentiated cultures. A conspicuous increase in the number and clustering of GSI-B4 lectin-binding cells, indicating a microglial response, was found at concentrations between 10(-10) M and 10(-7) M. These development-dependent and cell type-specific effects may reflect the pathogenic potential of long-term exposure to subclinical doses of mercury.

Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model.

An efficient screening strategy for the identification of potentially interesting low-abundance antifungal natural products in crude extracts that combines both a sensitive bioautography assay and high performance liquid chromatography (HPLC) microfractionation was developed. This method relies on high performance thin layer chromatography (HPTLC) bioautography with a hypersusceptible engineered strain of Candida albicans (DSY2621) for bioactivity detection, followed by the evaluation of wild type strains in standard microdilution antifungal assays. Active extracts were microfractionated by HPLC in 96-well plates, and the fractions were subsequently submitted to the bioassay. This procedure enabled precise localisation of the antifungal compounds directly in the HPLC chromatograms of the crude extracts. HPLC-PDA-mass spectrometry (MS) data obtained in parallel to the HPLC antifungal profiles provided a first chemical screening about the bioactive constituents. Transposition of the HPLC analytical conditions to medium-pressure liquid chromatography (MPLC) allowed the efficient isolation of the active constituents in mg amounts for structure confirmation and more extensive characterisation of their biological activities. The antifungal properties of the isolated natural products were evaluated by their minimum inhibitory concentration (MIC) in a dilution assay against both wild type and engineered strains of C. albicans. The biological activity of the most promising agents was further evaluated in vitro by electron microscopy and in vivo in a Galleria mellonella model of C. albicans infection. The overall procedure represents a rational and comprehensive means of evaluating antifungal activity from various perspectives for the selection of initial hits that can be explored in more in-depth mode-of-action studies. This strategy is illustrated by the identification and bioactivity evaluation of a series of antifungal compounds from the methanolic extract of a Rubiaceae plant, Morinda tomentosa, which was used as a model in these studies.

RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.

Plant- and microbe-derived compounds affect the expression of genes encoding antifungal compounds in a pseudomonad with biocontrol activity.

We have investigated the impacts of 63 different low-molecular-weight compounds, most of them plant derived, on the in vitro expression of two antifungal biosynthetic genes by the plant-protecting rhizobacterium Pseudomonas fluorescens CHA0. The majority of the compounds tested affected the expression of one or both antifungal genes. This suggests that biocontrol activity in plant-beneficial pseudomonads is modulated by plant-bacterium signaling.

Drug concentration response relationships in normal volunteers after oral administration of losartan, an angiotensin II receptor antagonist.

The aim of this study was to investigate the relationships between plasma concentrations of losartan, an orally active angiotensin II inhibitor, its active metabolite EXP3174, and angiotensin II blockade. Six healthy subjects received single oral doses of 40, 80, or 120 mg losartan and placebo at 1-week intervals in a crossover study. Angiotensin II blockade was assessed by the blood pressure response to exogenous angiotensin II before and after losartan administration. EXP3174 reached higher plasma concentrations and was eliminated more slowly than its parent compound; its levels paralleled the profile of angiotensin II blockade closer than losartan. Inhibition of the pressure response was dose dependent. The Hill-shaped relationship between response and EXP3174 concentration (or time-integrated variables) approached a plateau with 80 mg. The dose-dependent increase in plasma renin and angiotensin II exhibited a considerable individual scatter. We conclude that losartan produces a dose-dependent, effective angiotensin II blockade that is largely determined by the active metabolite EXP3174.

Comparative genome analysis of Pseudomonas knackmussii B13, the first bacterium known to degrade chloroaromatic compounds.

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103 kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a 'core' region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220 kb region and a prophage that drastically change the host metabolic capacity and survivability.

Anthracene-tethered ruthenium(II) arene complexes as tools to visualize the cellular localization of putative organometallic anticancer compounds.

Anthracene derivatives of ruthenium(II) arene compounds with 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (pta) or a sugar phosphite ligand, viz., 3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-d-glucofuranoside, were prepared in order to evaluate their anticancer properties compared to the parent compounds and to use them as models for intracellular visualization by fluorescence microscopy. Similar IC(50) values were obtained in cell proliferation assays, and similar levels of uptake and accumulation were also established. The X-ray structure of [{Ru(η(6)-C(6)H(5)CH(2)NHCO-anthracene)Cl(2)(pta)] is also reported.

Alcohol and cannabis use as risk factors for injury--a case-crossover analysis in a Swiss hospital emergency department.

BACKGROUND: There is sufficient and consistent evidence that alcohol use is a causal risk factor for injury. For cannabis use, however, there is conflicting evidence; a detrimental dose-response effect of cannabis use on psychomotor and other relevant skills has been found in experimental laboratory studies, while a protective effect of cannabis use has also been found in epidemiological studies. METHODS: Implementation of a case-crossover design study, with a representative sample of injured patients (N = 486; 332 men; 154 women) from the Emergency Department (ED) of the Lausanne University Hospital, which received treatment for different categories of injuries of varying aetiology. RESULTS: Alcohol use in the six hours prior to injury was associated with a relative risk of 3.00 (C.I.: 1.78, 5.04) compared with no alcohol use, a dose-response relationship also was found. Cannabis use was inversely related to risk of injury (RR: 0.33; C.I.: 0.12, 0.92), also in a dose-response like manner. However, the sample size for people who had used cannabis was small. Simultaneous use of alcohol and cannabis did not show significantly elevated risk. CONCLUSION: The most surprising result of our study was the inverse relationship between cannabis use and injury. Possible explanations and underlying mechanisms, such as use in safer environments or more compensatory behavior among cannabis users, were discussed.

Comparative genome analysis of Pseudomonas knackmussii B13, the first bacterium known to degrade chloroaromatic compounds.

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103 kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a 'core' region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220 kb region and a prophage that drastically change the host metabolic capacity and survivability.

Trials using a crossover design and ambulatory blood pressure recordings to determine the efficacy of antihypertensive agents in individual patients.

The antihypertensive effects of the beta-blocking agent betaxolol and the calcium entry blocker verapamil were compared in a crossover single-blind trial. Seventeen patients with uncomplicated essential hypertension took either betaxolol or a slow-release formulation of verapamil for two consecutive 6-week periods. The sequence of treatment phases was randomly allocated and a 2-week washout period preceded each treatment. The antihypertensive effect of the test drugs was assessed both at the physician's office and during everyday activities using a portable blood pressure recorder. The crossover design of the trial made it possible to evaluate the antihypertensive efficacy of betaxolol and verapamil both in the group as a whole and in the individual patient. The individual patient response to one of these agents was not a reliable indicator of the same patient's response to the alternative agent. Betaxolol brought both office and ambulatory recorded blood pressures under control in a larger fraction of patients than verapamil, although the magnitude of the blood pressure fall in the responders was equal for each drug. These observations stress the need for an individualized approach to the evaluation of antihypertensive therapy. The present results also demonstrate that optimal antihypertensive therapy is still a matter of trial and error. The precise methodology that ought to characterize crossover trials may make it possible to improve the therapeutic approach to hypertensive patients.

Effect of water velocity on the uptake of polychlorinatedd biphenyls (PCBs) by silicone rubber (SR) and low-density polyethylene (LDPE) passive samplers: an assessment of the efficiency of performance reference compounds (PRCs) in river-like flow conditions

One aim of this study is to determine the impact of water velocity on the uptake of indicator polychlorinated biphenyls (iPCBs) by silicone rubber (SR) and low-density polyethylene (LDPE) passive samplers. A second aim is to assess the efficiency of performance reference compounds (PRCs) to correct for the impact of water velocity. SR and LDPE samplers were spiked with 11 or 12 PRCs and exposed for 6 weeks to four different velocities (in the range of 1.6 to 37.7 cm s−1) in river-like flow conditions using a channel system supplied with river water. A relationship between velocity and the uptakewas found for each iPCB and enables to determine expected changes in the uptake due to velocity variations. For both samplers, velocity increases from 2 to 10 cm s−1, 30 cm s−1 (interpolated data) and 100 cm s−1 (extrapolated data) lead to increases of the uptake which do not exceed a factor of 2, 3 and 4.5, respectively. Results also showed that the influence of velocity decreased with increasing the octanol-water coefficient partition (log Kow) of iPCBs when SR is used whereas the opposite effect was observed for LDPE. Time-weighted average (TWA) concentrations of iPCBs in water were calculated from iPCB uptake and PRC release. These calculations were performed using either a single PRC or all the PRCs. The efficiency of PRCs to correct the impact of velocity was assessed by comparing the TWA concentrations obtained at the four tested velocities. For SR, a good agreement was found among the four TWA concentrations with both methods (average RSD b 10%). Also for LDPE, PRCs offered a good correction of the impact of water velocity (average RSD of about 10 to 20%). These results contribute to the process of acceptance of passive sampling in routine regulatory monitoring programs.

Antifungal activity of compounds targeting the Hsp90-calcineurin pathway against various mould species.

OBJECTIVES: Invasive mould infections are associated with a high mortality rate and the emergence of MDR moulds is of particular concern. Calcineurin and its chaperone, the heat shock protein 90 (Hsp90), represent an important pathway for fungal virulence that can be targeted at different levels. We investigated the antifungal activity of compounds directly or indirectly targeting the Hsp90-calcineurin axis against different mould species. METHODS: The in vitro antifungal activity of the anticalcineurin drug FK506 (tacrolimus), the Hsp90 inhibitor geldanamycin, the lysine deacetylase inhibitor trichostatin A and the Hsp70 inhibitor pifithrin-μ was assessed by the standard broth dilution method against 62 clinical isolates of Aspergillus spp. and non-Aspergillus moulds (Mucoromycotina, Fusarium spp., Scedosporium spp., Purpureocillium/Paecilomyces spp. and Scopulariopsis spp.) RESULTS: FK506 had variable antifungal activity against different Aspergillus spp. and was particularly active against Mucor spp. Geldanamycin had moderate antifungal activity against Fusarium spp. and Paecilomyces variotii. Importantly, trichostatin A had good activity against the triazole-resistant Aspergillus ustus and the amphotericin B-resistant Aspergillus terreus as well as the MDR Scedosporium prolificans. Moreover, trichostatin A exhibited synergistic interactions with caspofungin against A. ustus and with geldanamycin against Rhizopus spp. for which none of the other agents showed activity. Pifithrin-μ exhibited little antifungal activity. CONCLUSIONS: Targeting the Hsp90-calcineurin axis at different levels resulted in distinct patterns of susceptibility among different fungal species. Lysine deacetylase inhibition may represent a promising novel antifungal strategy against emerging resistant moulds.