We investigated Legionella and Pseudomonas contamination of hot water in a cross-sectional multicentric survey in Italy. Chemical parameters (hardness, free chlorine, and trace elements) were determined. Legionella spp. were detected in 33 (22.6%) and Pseudomonas spp. in 56 (38.4%) of 146 samples. Some factors associated with Legionella contamination were heater type, tank distance and capacity, water plant age, and mineral content. Pseudomonas presence was influenced by water source, hardness, free chlorine, and temperature. Legionella contamination was associated with a centralized heater, distance from the heater point >10 m, and a water plant >10 years old. Furthermore, zinc levels of <20 μg/L and copper levels of >50 μg/L appeared to be protective against Legionella colonization. Legionella species and serogroups were differently distributed according to heater type, water temperature, and free chlorine, suggesting that Legionella strains may have a different sensibility and resistance to environmental factors and different ecologic niches.

A cross-sectional multicenter survey of Italian hotels was conducted to investigate Legionella spp. contamination of hot water. Chemical parameters (hardness, free chlorine concentration, and trace element concentrations), water systems, and building characteristics were evaluated to study risk factors for colonization. The hot water systems of Italian hotels were strongly colonized by Legionella; 75% of the buildings examined and 60% of the water samples were contaminated, mainly at levels of >10 3 CFU liter ؊1 , and Legionella pneumophila was the most frequently isolated species (87%). L. pneumophila serogroup 1 was isolated from 45.8% of the contaminated sites and from 32.5% of the hotels examined. When a multivariate logistic model was used, only hotel age was associated with contamination, but the risk factors differed depending on the contaminating species and serogroup. Soft water with higher chlorine levels and higher temperatures were associated with L. pneumophila serogroup 1 colonization, whereas the opposite was observed for serogroups 2 to 14. In conclusion, Italian hotels, particularly those located in old buildings, represent a major source of risk for Legionnaires' disease due to the high frequency of Legionella contamination, high germ concentration, and major L. pneumophila serogroup 1 colonization. The possible role of chlorine in favoring the survival of Legionella species is discussed.

By using high-density oligonucleotide arrays, we profiled gene expression in reward-related brain regions of rats that developed escalated cocaine intake after extended access to cocaine (6 h per day). Rats allowed restricted daily access to cocaine (only 1 h) that displayed a stable level of cocaine intake and cocaine naive rats were used for controls. Four analysis methods were compared: Affymetrix MICROARRAY SUITE 4 and MICROARRAY SUITE 5, which use perfect-match-minus-mismatch models, and DCHIP and RMA, which use perfect-match-only models to generate expression values. Results were validated by RT-PCR in individual animals from an independent replication of the experiment. A small number of genes was associated with escalated cocaine intake (ESC genes). Unexpectedly, of the brain regions examined [prefrontal cortex, nucleus accumbens, septum, lateral hypothalamus (LH), amygdala, and ventral tegmental area], the LH was the most transcriptionally responsive in escalation of cocaine intake. Most of the ESC genes identified are also expressed during synaptogenesis and synaptic plasticity and include genes that code for several presynaptic and postsynaptic proteins involved in neurotransmission. These results suggest that LH intrinsic circuitry undergoes a structural reorganization during escalation of cocaine use. This remodeling of LH circuitry could contribute to the chronic deficit in reward function that has been hypothesized to drive the transition to drug addiction. Results also support the value of using multiple analysis strategies to identify the most robust changes in gene expression and to compensate for the biases that affect each strategy.release machinery ͉ ␦-catenin ͉ Glu receptor-interacting protein 2 ͉ PKC␥ ͉ fractalkine A current challenge for the neuroscience of drug addiction is to understand the molecular mechanisms responsible for the development of compulsive drug use (1). Such a transition is generally associated with a pattern of escalating drug use whereby consumption increases over time and becomes increasingly difficult to control. In the present study, we investigated gene expression changes associated with drug addiction by using an animal model that demonstrates escalation of cocaine intake. In this model, drug intake gradually escalates when daily access to the drug is increased to Ն6 h [long access (LgA)] (2, 3), whereas with only 1 h of access per day [short access (ShA)], drug intake remains low and stable over time. The difference in cocaine consumption between ShA and LgA animals has been hypothesized to model the difference drawn by clinicians between controlled and compulsive drug use (2, 3). This view is supported by recent results showing that escalation in cocaine intake in LgA animals is associated with an increased motivation to seek and to take cocaine and with a persistent downregulation of brain reward function when compared with ShA animals (3-5). Therefore, escalation of cocaine intake in LgA rats appears to replicate the behavioral and neuroadaptive changes assoc...

Aims: A simple and rapid method was described for DNA isolation directly from activated sludge or other environmental sources, including soil and sediments. Methods and Results: The present method is based on microwave thermal shock and provides DNA suitable for further analysis. It is also effective for RNA extraction. Conclusions: The protocol is effective, easy, fast and does not require the use of expensive equipment or reagents. Significance and Impact of the Study: The described method can be applied to difficult substrates in environmental microbiology studies.

We have ectopically expressed transcription factor ETS1 in two different highly tumorigenic human colon cancer cell lines, DLD- (25).Isolation of cDNA Coding for Mutant ETS1. Total genomic DNA from DE1-2-3 cells (1.2 ,ug) was used for polymerase chain reaction (PCR) with sense (5'-ggattcCACCATGAAG-GCGGCCGT-3'; positions -4 to 14) and antisense (5'-caagcttgTCAGTGCCATCACTC-3'; positions 1321-1335) ETS1 primer pairs to clone integrated ETS1 cDNA as described (26).Cell Culture and Transfection. The human colon carcinoma cell lines DLD-1 (27) and HCT116 (28) were transfected with human ETS1 cDNA expression vectors along with pSV2neo by use of Lipofectin reagent (GIBCO/BRL). Transfectants were selected with G418 (400 ,ug/ml). DE-1-1, DE1-1-7, and DE1-3-2 are DLD-1 transfectants obtained with pcDM7 ETS1 expression vector, whereas DE1-2-3, DE1-2-4, and DE1-2-8 are DLD-1 transfectants obtained with pcDNAI ETS1 expression vector. These were done as described (7,9,(29)(30)(31). RESULTS AND DISCUSSIONGeneration of DLD-1 Cells Expressing ETS1 Proteins. We expressed the full-length ETS1 protein in DLD-1 and HCT116 cells under the control of the cytomegalovirus promoter and obtained several independent cell lines. These cell lines were derived from single cell clones. DNA blot analyses of transfectants digested with multiple restriction enzymes confirmed that these transfectants truly represented independent clones (data not shown). ETS1 protein formation was examined by radioimmunoprecipitation using ETS1-specific monoclonal antibody (13,31). In DLD-1 transfectants (DE1-1-1, DE1-1-7, Abbreviations: CAT, chloramphenicol acetyltransferase; EBS, ETS1-binding sequence. tPresent address:

A growing number of people attend swimming facilities for recreational activities, rehabilitative treatments, or sport. Filamentous fungi and yeast can be isolated from contaminated air, water and surfaces and may represent a biological risk for employees and users. Here we investigated the occurrence of mycotic species, in a sample of Italian swimming pools (n = 10). Detection and identification of isolated species were achieved by cultural and morphological methods. Results revealed moderate mycotic titres and a high biodiversity. Penicillium spp., Aspergillus spp., Cladosporium spp. and Alternaria sp., were constantly detected in air and surfaces sampled by the swimming area, while pathogenic yeast Candida albicans was never detected. Fusarium spp. was the most common taxon isolated from surfaces. For one facility, we typed the genotypic profiles and studied, by genetic typing, the spatial and temporal distribution of isolates. Phylogenetic relationships between species were analysed by alignment of small ribosomal subunit RNA sequences.

Real-time PCR represents an effective approach for typing ACTN3 alleles. Allelic frequencies in the Italian population are consistent with those seen in other studies on Caucasians.

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