Mycoremediation effect of Trichoderma harzianum strain T22 combined with ozonation in diesel-contaminated sand.

This study aimed to determine the ability of the fungus Trichoderma harzianum strain T22 (Th-T22) to utilize diesel fuel as a carbon source. The potential use of Th-T22 for diesel bioremediation in an artificial soil was tested by inoculating a diesel-sand mixture with a fungal mycelial suspension of Th-T22. Given the ability of ozone to degrade compounds with low biochemical reactivity, the effect of a pre- and post-ozonation was also evaluated. The survival, growth and sporulation of Th-T22 throughout the bioremediation trial were monitored in all the treatments. In the post-ozonation treatments, the biodegradation percentages of diesel removal were 70.16% and 88.35% in Th-T22-inoculated sand treated or untreated with the antibacterial streptomycin, respectively. The results showed that ozonation alone caused good removal efficiencies (41.9%) but it was much more effective if combined with Th-T22 in a post-ozonation regime, whereas pre-ozonation negatively affected the subsequent biodegradation, likely due to its disinfectant and oxidizing effect on Th-T22. The results obtained demonstrated the significant mycoremediation ability of Th-T22 in diesel-contaminated sand and its possible use as a bioremediation agent for diesel spills in polluted sites.

Case of a patient with heart failure, dilated cardiomyopathy and atrial fibrillation treated with sacubitril/valsartan.

A novel combination consisting of the neprilysin inhibitor, sacubitril, and the angiotensin-receptor blocker, valsartan (belonging to the newly established class of angiotensin receptor/neprilysin inhibitors), was shown to be effective in the treatment of heart failure (HF) by improving patient clinical status, and reducing re-hospitalization rate and mortality. We report a case of a 29 year old male with HF, dilated cardiomyopathy possibly related to myocarditis and atrial fibrillation with reduced ejection fraction. Sacubitril/valsartan treatment was initiated after two years of standard treatment. In two years, therapy with sacubitril/valsartan led to persistence in sinus rhythm, progressive recovery of ejection fraction, functionality and reduction of cardiac volumes. The patient is currently in good condition and has suspended diuretic therapy in the last six months.

CO2 and N2O from water resource recovery facilities: Evaluation of emissions from biological treatment, settling, disinfection, and receiving water body.

Water resource recovery facilities (WRRFs) contribute to climate change and air pollution, as they are anthropogenic potential sources of direct and indirect emission of greenhouse gases (GHGs). Studies concerning the monitoring and accounting for GHG emissions from WRRFs are of increasing interest. In this study, the floating hood technique for gas collection was coupled with the off-gas method to monitor and apportion nitrous oxide (N2O) and carbon dioxide (CO2) emissions from both aerated and non-aerated tanks in a municipal water resource recovery facility, in order to investigate its carbon footprint (CFP). To our knowledge, this is the first time that the chamber technique was applied to evaluate gas fluxes from the settler, where an emission factor (EF) of 4.71 ∗ 10-5 kgCO2,eq kgbCOD-1 was found. Interesting results were found in the disinfection unit, which was the major contributor to direct N2O emissions (with a specific emission factor of 0.008 kgCO2,eq kgbCOD-1), due to the chemical interaction between hydroxylamine and the disinfectant agent (hypochlorite). The specific emission factor of the biological aerated tank was 0.00112 kgCO2,eq kgbCOD-1. The average direct CO2 emission was equal to 0.068 kgCO2 kgbCOD-1 from the activated sludge tank and to 0.00017 kgCO2 kgbCOD-1 from the secondary clarifier. Therefore, taking into account the contribution of both direct N2O and CO2 emissions, values of 0.069 kgCO2,eq kgbCOD-1, 0.008 kgCO2,eq kgbCOD-1 and 0.00022 kgCO2,eq kgbCOD-1, were found for the net CFP of the aerated compartment, the disinfection unit and the clarifier, respectively. The plant energy Footprint (eFP) was also evaluated, confirming that the aeration system is the major contributor to energy consumption, as well as to indirect CO2 emission, with a specific eFP of 1.49 kWh kgbCOD-1.

Biological monitoring of occupational exposure to N,N-dimethylacetamide with identification of a new metabolite.

AIMS: To study the concentration of N,N-dimethylacetamide (DMA) and its metabolite, N-methylacetamide (NMA), in urine of workers occupationally exposed to DMA in a factory producing synthetic acrylic fibres. METHODS: During the first phase, 223 workers exposed to low environmental concentrations of DMA provided urine samples at the end of a work shift. High concentrations of the unmodified solvent and its metabolite were found in a group of workers whose job was to start up machinery. The second and third phases focused on conditions favouring high uptake of DMA. RESULTS: The highest concentrations of unmodified solvent and NMA were found in the urine of workers recently engaged in starting up machinery. NMA in urine was 1.5-173.6 mg/g creatinine (median 20.5). In spite of the low environmental concentration, about 20% of the urine concentration of NMA was higher than 30 mg/g creatinine. Dermal absorption of DMA was high. A shower and a change of clothing at the end of the work shift, and washing away any solvent left on the skin, ensured that dermal absorption of DMA did not continue. This significantly reduced the NMA urinary concentration at values lower than 30 mg/g creatinine. In some urine samples, S-acetamidomethyl-mercapturic acid was identified by NMR analysis; this is probably a metabolite of N,N-dimethylacetamide--it has never before been identified in humans or animals. CONCLUSIONS: Even at low environmental concentrations of DMA, dermal absorption can be considerable. Unmodified DMA and NMA concentrations in urine are good biomarkers for monitoring occupational exposure to the solvent.

The induction of cyclooxygenase-2 mRNA in macrophages is biphasic and requires both CCAAT enhancer-binding protein beta (C/EBP beta ) and C/EBP delta transcription factors.

Prostaglandins are important mediators of activated macrophage functions, and their inducible synthesis is mediated by cyclooxygenase-2 (COX-2). Here, we make use of the murine macrophage cells RAW264 as well as of immortalized macrophages derived from mice deficient for the transcription factor CCAAT enhancer-binding protein beta (C/EBP beta) to explore the molecular mechanisms regulating COX-2 induction in activated macrophages. We demonstrate that lipopolysaccharide-mediated COX-2 mRNA induction is biphasic. The initial phase is independent of de novo protein synthesis, correlates with cAMP-response element-binding protein (CREB) activation, is inhibited by treatments that abolish CREB phosphorylation and reduce NF-kappa B-mediated gene activation, and requires the presence of the transcription factor C/EBP beta. On the other hand, C/EBP delta appears to be essential in addition to C/EBP beta to effect the second phase of COX-2 gene transcription, which is important for maintaining the induced state and requires de novo protein synthesis. Indeed, both phases of COX-2 induction were defective in C/EBP beta-/- macrophages. Moreover, the synthesis of C/EBP delta was increased dramatically by treatment with lipopolysaccharide and, like COX-2 induction, repressed by combined inhibition of the MAPK and of the SAPK2/p38 cascades. Taken together, these data identify CREB, NF-kappa B, and both C/EBP beta and -delta as key factors in coordinately orchestrating transcription from the COX-2 promoter in activated macrophages.

The transcription factor C/EBPbeta is essential for inducible expression of the cox-2 gene in macrophages but not in fibroblasts.

Cyclooxygenase-2 (COX-2) is the rate-limiting enzyme for the inducible synthesis of prostaglandins, and its up-regulated activity is thought to play a pathological role in diseases such as inflammatory bowel disease, rheumatoid arthritis, and cancer. Regulation of COX-2 expression is complex and appears to involve diversified mechanisms in different cell types and conditions. Here we make use of immortalized macrophages and fibroblasts that we have generated from C/EBPbeta-deficient mice to directly test and compare the specific role played by this factor in inducible COX-2 expression in these two cell types. We could demonstrate that COX-2 mRNA induction and promoter activity were profoundly impaired in C/EBPbeta(-/-) macrophages and could be rescued by expression of C/EBPbeta. The obligatory role of C/EBPbeta in COX-2 expression appeared to be mediated exclusively by the C/EBP element located at positions -138/-130 of the murine cox-2 promoter, and did not involve altered activity at the level of the other promoter elements described previously (the -402/-392 NF-kappaB site, the -59/-48 CRE/E box element, and a potential second C/EBP site located at positions -93/-85). In contrast, COX-2 induction was completely normal in C/EBPbeta-deficient fibroblasts, thus highlighting the diversity of cell-specific molecular mechanisms in determining inducible COX-2 expression and prostaglandins production.

Specificity and mechanism of action of some commonly used protein kinase inhibitors.

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

Role of mitogen-activated protein kinase cascades in mediating lipopolysaccharide-stimulated induction of cyclooxygenase-2 and IL-1 beta in RAW264 macrophages.

LPS stimulation of RAW264 macrophages triggered the activation of mitogen- and stress-activated protein kinases-1 and -2 (MSK1, MSK2) and their putative substrates, the transcription factors cyclic AMP response element-binding protein (CREB) and activating transcription factor-1 (ATF1). The activation of MSK1/MSK2 was prevented by preincubating the cells with a combination of two drugs that suppress activation of the classical mitogen-activated protein kinase cascade and stress-activated protein kinase/p38, respectively, but inhibition was only partial in the presence of either inhibitor. The LPS-stimulated activation of CREB and ATF1, the transcription of the cyclooxygenase-2 (COX-2) and IL-1 beta genes (the promoters of which contain a cyclic AMP response element), and the induction of the COX-2 protein were prevented by the same drug combination, as well as by Ro 318220 or H89, potent inhibitors of MSK1/MSK2. Two other transcription factors, C/EBP beta and NF-kappa B, have been implicated in the transcription of the COX-2 gene. However, PD 98059 and/or SB 203580 did not prevent the LPS-induced increase in the level of the transcription factor C/EBP beta, and none of the four inhibitors used in this study prevented the activation of NF-kappa B. Our results demonstrate that two different mitogen-activated protein kinase cascades are rate limiting for the LPS-induced activation of CREB/ATF1 and the transcription of the COX-2 and IL-1 beta genes. They also suggest that MSK1 and MSK2 may play a role in these processes and hence are potential targets for the development of novel antiinflammatory drugs.

Role of MAP kinase cascades in inducing arginine transporters and nitric oxide synthetase in RAW264 macrophages.

Bacterial lipopolysaccharide (LPS) in the presence of interferon gamma (IFNgamma) stimulates the synthesis of the cationic amino acid transporter 2B (CAT-2B) and inducible nitric oxide synthetase (iNOS) in RAW264 macrophages, which are thought to underlie the increased rate of arginine uptake into these cells and its conversion to nitric oxide, respectively. Here I demonstrate that the LPS- and IFNgamma-induced increase in arginine uptake into RAW264 cells is partially suppressed in the presence of PD 98059, partially suppressed in the presence of SB 203580, and completely inhibited by both drugs. In contrast, the LPS- and IFNgamma-induced synthesis of CAT-2B mRNA and iNOS protein is unaffected by PD 98059 and SB 203580. The results indicate that the MAPK/ERK and SAPK2/p38 cascades are both rate-limiting for LPS- and IFNgamma-stimulated arginine uptake, but not for iNOS synthesis. They also suggest that PD 98059 and SB 203580 suppress CAT-2B synthesis at a post-transcriptional level.

Presenilin-1 is processed into two major cleavage products in neuronal cell lines.

Presenilin-1 (PS-1) has been identified as the protein encoded by the chromosome 14 locus that, when mutated, leads to familial Alzheimer's disease (FAD). Using PS-1 transfected SHSY5Y neuroblastoma cells, we have demonstrated by immunodetection, using polyclonal antibodies, that PS-1 is processed to give two fragments: an N-terminal 28 kDa fragment, and a C-terminal 18 kDa fragment. In a number of non-transfected cell types, most PS-1 is detected as the cleaved products. The molecular weights of the PS-1 cleavage products suggest that the cleavage point will most probably be within a region of the hydrophilic loop domain coded for by either exon 8 or 9 of the PS-1 gene. The clustering of FAD mutations within exon 8 strongly suggests that it encodes a key functional domain. It seems likely that the cleavage of PS-1 is crucial to some aspect of its functionality. An understanding of this process will give insights into the pathology of AD, and may offer new opportunities for therapeutic intervention.

Effects of fluvoxamine on the protein phosphorylation system associated with rat neuronal microtubules.

We have studied the phosphorylation system associated with the rat cerebrocortical microtubule fraction after short- and long-term administration (15 mg/kg) of fluvoxamine, a selective serotonin reuptake inhibitor with antidepressant activity. Fluvoxamine administered for 5 days significantly enhanced the 32P incorporation stimulated by cAMP into MAP2, while it failed to produce this effect after 12 and 21 days. Moreover, in the same periods of treatment no changes were observed in basal phosphorylation and in the pattern of microtubule proteins. In conclusion, our results suggest that changes in the protein phosphorylation system associated with the microtubule fraction could represent an early neurochemical modification involved in the action of fluvoxamine.