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To analyze possible effects of microwaves on gene expression, mice were exposed to global system for mobile communication (GSM) 1800 MHz signal for 1 h at a whole body SAR of 1.1 W/kg. Gene expression was studied in the whole brain, where the average SAR was 0.2 W/kg, by expression microarrays containing over 22,600 probe sets. Comparison of data from sham and exposed animals showed no significant difference in gene expression modulation. However, when less stringent constraints were adopted to analyze microarray results, 75 genes were found to be modulated following exposure. Forty-two probes showed fold changes ranging from 1.5 to 2.8, whereas 33 were down-regulated from 0.67- to 0.29-fold changes, but these differences in gene expression were not confirmed by real-time PCR. Under these specific limited conditions, no consistent indication of gene expression modulation in whole mouse brain was found associated to GSM 1800 MHz exposure.

PURPOSE: Development of two novel sets of primers and probes to detect R577X and Q523R polymorphisms of the α-actinin-3 (ACTN3) gene by real-time PCR. We report the allelic frequencies observed in Italian individuals from the general population and athletes. Athletic performance is influenced by training, environmental factors, and genetic predisposition. Actn3 belongs to a family of actin-binding proteins and is supposed to influence sport performance. METHODS: Primer-probe set design and protocol optimization for real-time PCR genotyping of R577X and Q523R polymorphisms. The assay was verified using a traditional PCR-RFLP approach and applied on an Italian population sample (102 male subjects and 42 athletes). RESULTS: Haplotype distribution confirmed the presence of linkage disequilibrium between the polymorphisms, both in the Italian general population and athletes (respectively: χ = 54.4, P ≤ 0.001 and χ = 24.5, P ≤ 0.001). Within the general population, a large percentage of homozygous subjects (21.6%) was deficient for Actn3. No significant differences were observed in athletes. The concordance between PCR-RFLP and real-time PCR results was 100and 93% for polymorphisms Q523R and R577X, respectively. CONCLUSION: Real-time PCR represents an effective approach fortyping ACTN3 alleles. Allelic frequencies in the Italian population are consistent with those seen in other studies on Caucasians.

Administration of prohibited substances to enhance athletic performance represents an emerging medical, social, ethical and legal issue. Traditional controls are based on direct detection of substances or their catabolites. However out-of-competition doping may not be easily revealed by standard analytical methods. Alternative indirect control strategies are based on the evaluation of mid- and long-term effects of doping in tissues. Drug-induced long-lasting changes of gene expression may be taken as effective indicators of doping exposure. To validate this approach, we used real-time PCR to monitor the expression pattern of selected genes in human haematopoietic cells exposed to nandrolone, insulin-like growth factor I (IGF-I) or growth hormone (GH). Some candidate genes were found significantly and consistently modulated by treatments. Nandrolone up-regulated AR, ESR2 and PGR in K562 cells, and SRD5A1, PPARA and JAK2 in Jurkat cells; IGF-I up-regulated EPOR and PGR in HL60 cells, and SRD5A1 in Jurkat; GH up-regulated SRD5A1 and GHR in K562. GATA1 expression was down-regulated in IGF-1-treated HL60, ESR2 was down-regulated in nandrolone-treated Jurkat, and AR and PGR were down-regulated in GH-treated Jurkat. This pilot study shows the potential of molecular biology-based strategies in anti-doping controls.

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.

The cauliflower mosaic virus 35S promoter (CaMV35s) is extensively used in genetically modified crops for human and animal consumption. Horizontal gene transfer is attracting particular attention, in light of experimental reports, showing the presence of dietary DNA into animal tissues. Health implications may derive from possible activities of the heterologous promoter in mammalian cells after integration in the host genome. To evaluate this hypothesis, in vivo and in vitro experiments were performed using GFP as reporter gene. Recombinant plasmid DNA was fed to Balb/c mice and searched in several tissues by PCR amplification. The activity of the plant virus promoter was assessed by RT-PCR and fluorescence microscopy after liposome-mediated transfection of murine gonadic cells. Obtained data did not highlight evidences of dietary DNA transfer in mice. No CaMV35s transcriptional activity was detected in this experimental model. These findings emphasize the need for further studies and standardized methods.

Sequence-based approaches to prokaryotic systematics and typing represent a modern and promising strategy in epidemiology and environmental microbiology. GenEnv, a database-driven system for bacterial typing, was developed in order to provide user friendly tools for supporting biomolecular analysis of bacteria. The family Vibrionaceae represents a heterogeneous taxon of aquatic microrganisms, harbouring a plethora of genomes currently analyzed by different molecular techniques. Under the query "Vibrio", GenEnv retrieved 256 organisms, included in a total number of 19 families. Overall, 548 sequences, comprising 16S rRNA (n = 402), rpoB (n = 1), gyrB (n = 145) were available. In addition, GenEnv system allowed primer design, homology analysis and restriction maps, for immediate applications to the study of Vibrionaceae.

OBJECTIVES: To perform a 3-year, prospective surveillance program for legionnaires disease (LD) in a large university hospital in Rome, and to assess the usefulness of the hospital water monitoring program in predicting the risk of nosocomial LD. METHODS: Samples from patients with new cases of nosocomial pneumonia were sent for legionella laboratory investigations. Meanwhile, water samples for bacteriological analysis were collected every 6 months from high- and medium-risk hospital wards (10 in total). Legionella pneumophila isolates collected were serotyped and analyzed by pulsed-field gel electrophoresis. RESULTS: From June 2001 through May 2004, the pneumonia surveillance identified one case of nosocomial LD among 43 cases of nosocomial pneumonia (2.3%). Environmental investigations detected L. pneumophila in 12 (18.7%) of the 64 water samples, of which 50% belonged to serogroup 1. The L. pneumophila count and the percentage of positive locations never exceeded 10(2) colony-forming units/L and 20%, respectively, except when the LD nosocomial case occurred (positive water samples, 40%; L. pneumophila count, <10(2) colony-forming units/L). Genotyping showed 3 prevalent clones of L. pneumophila in the water distribution network, of which one persisted over the 3 years. One clone contained 3 different L. pneumophila serogroups (2, 4, and 6). CONCLUSIONS: The low incidence of nosocomial cases of LD appears to be associated with a low percentage (<20%) of positive water samples per semester and with a low contamination level (<10(2) colony-forming units/L). An infection control system for nosocomial LD should, therefore, be based on both environmental and clinical surveillance, together with the appropriate maintenance of the hospital water distribution system.

In the present study we use modified CDR3 beta-chain spectratyping (immunoscope) to dissect the effect of Mycobacterium Tuberculosis -derived proteins on individual PLP139-151 specific cells in the SJL mouse strain. In this model, the immunoscope technique allows the characterization of a public TCR that involves rearrangement of Vbeta10 and Jbeta1.1 and a semiprivate TCR characterized by rearrangement of Vbeta4 and Jbeta 1.6. Both rearrangements are specific for PLP139-151and sequences of the CDR3 region of the two beta-chains show a conserved motif for the public rearrangement and related but more variable sequences for the semiprivate rearrangement. Mycobacterium Tuberculosis -derived proteins promote increase of IFN-gamma secreting cells. However, we observe that presence and amount of Mycobacterium Tuberculosis used during immunization have no effect on the frequency of usage, polarization and in vivo expansion of cells carrying the studied rearrangements. Rather, the strong Th1 promoting effect of adjuvant is possibly due to recruitment towards Th1 of a wider spectrum of TCR repertoires. Therefore, instead of having a comprehensive effect on the entire repertoire, Mycobacterium Tuberculosis modulates the immune response by affecting a subset of antigen-specific T cells whose polarization can be adapted to the environment. This step establishes the final balance between Th1 and Th2 and may be essential for the enhancement or protection of disease.

Species identification represents a critical issue in food chain safety and quality control. Several procedures are available to detect animal proteins in cattle feed or to trace transgenic foods. The most effective approach is based on the use of DNA as a marker. Amplification of DNA provides rapid, sensitive and specific protocols. Several target genes can be used, but new insights come from the mitochondrial genome, which is naturally amplified in each cell and shows a remarkable resistance to degradation. These are key points when analysing complex matrices such as foods, animal feedstuff or environmental samples. Traceability is important to prevent BSE or to monitor novel foods, such as genetically modified organisms. Amplification is commonly performed, but it requires expertise and a molecular biology laboratory to perform restriction analysis, electrophoresis or gel staining for the visualisation of results. Hereby, we consider a strategy based on multiple nested amplification and reverse hybridisation assay that virtually requires only a thermocycler and a water bath. The protocol is rapid and simple and can simultaneously detect different species in a DNA sample. This promising approach allows microarray developments, opening up to further perspectives. An international application has been published under the patent cooperation treaty. Presently, a ban on feeding ruminants on cattle-derived proteins is in force in Europe and USA. The identification of metazoan traces in a sample is not only a mere preventive measure for BSE, but represents a possible screening system for monitoring biotechnology products and procedures, as well as a quality control strategy to assure consumer's rights.

Giardia and Cryptosporidium are parasitic protozoa responsible for enteritis in man, often severe in children and in immunocompromised patients. Their transmissive stages, cysts and oocysts, voided in large number with the faeces of infected hosts, may contaminate sewage effluents and surface water. Detection of these microorganisms in water environmental samples is a critical issue. Conventional methods for detection of Cryptosporidium parvum and Giardia lamblia are labor-intensive and time-consuming. Molecular detection methods based on amplification techniques have been applied for water quality management, but require a previous protozoa enrichment step by filtration and elution. Direct environmental DNA extraction plays a key role, but is limited by copurification of polymerase inhibitors. We have compared lysis and DNA isolation methods for protozoa detection by real-time per. The molecular approach provides a promising and effective tool for flanking the traditional optical microscopy based assays.

Genetically Modified Organisms (GMOs) are a fact of modern agriculture and a major field of discussion in biotechnology. As science incessantly achieves innovative and unexpected breakthroughs, new medical, political, ethical and religious debates arise over the production and consumption of transgenic organisms. Despite no described medical condition being directly associated with a diet including approved GM crops in large exposed populations such as 300,000,000 Americans and a billion Chinese, public opinion seems to look at this new technology with either growing concern or even disapproval. It is generally recognized that a high level of vigilance is necessary and highly desirable, but it should also be considered that GMOs are a promising new challenge for the III Millennium societies, with remarkable impact on many disciplines and fields related to biotechnology. To acquire a basic knowledge on GMO production, GM-food consumption, GMO interaction with humans and environment is of primary importance for risk assessment. It requires availability of clear data and results from rigorous experiments. This review will focus on public health risks related with a GMO-containing diet. The objective is to summarize state of the art research, provide fundamental technical information, point out problems and perspectives, and make available essential tools for further research. Are GMO based industries and GMO-derived foods safe to human health? Can we consider both social, ethical and public health issues by means of a constant and effective monitoring of the food chain and by a clear, informative labeling of the products? Which are the so far characterized or alleged hazards of GMOs? And, most importantly, are these hazards actual, potential or merely contrived? Several questions remain open; answers and solutions belong to science, to politics and to the personal opinion of each social subject.