Spatio-temporal variability of airborne bacterial communities and their correlation with particulate matter chemical composition across two urban areas.

The study of spatio-temporal variability of airborne bacterial communities has recently gained importance due to the evidence that airborne bacteria are involved in atmospheric processes and can affect human health. In this work, we described the structure of airborne microbial communities in two urban areas (Milan and Venice, Northern Italy) through the sequencing, by the Illumina platform, of libraries containing the V5-V6 hypervariable regions of the 16S rRNA gene and estimated the abundance of airborne bacteria with quantitative PCR (qPCR). Airborne microbial communities were dominated by few taxa, particularly Burkholderiales and Actinomycetales, more abundant in colder seasons, and Chloroplasts, more abundant in warmer seasons. By partitioning the variation in bacterial community structure, we could assess that environmental and meteorological conditions, including variability between cities and seasons, were the major determinants of the observed variation in bacterial community structure, while chemical composition of atmospheric particulate matter (PM) had a minor contribution. Particularly, Ba, SO4 (2-) and Mg(2+) concentrations were significantly correlated with microbial community structure, but it was not possible to assess whether they simply co-varied with seasonal shifts of bacterial inputs to the atmosphere, or their variation favoured specific taxa. Both local sources of bacteria and atmospheric dispersal were involved in the assembling of airborne microbial communities, as suggested, to the one side by the large abundance of bacteria typical of lagoon environments (Rhodobacterales) observed in spring air samples from Venice and to the other by the significant effect of wind speed in shaping airborne bacterial communities at all sites.

Development of microbial engineered whole-cell systems for environmental benzene determination.

This paper reports the development of two recombinant bacterial systems that can be used to monitor environmental benzene contamination based on Escherichia coli, which carry genes coding for benzene dioxygenase and benzene dihydrodiol dehydrogenase from Pseudomonas putida MST. E. coli strains express these two enzymes under the control of the Ptac promoter or without any induction. These activities can be detected electrochemically or colorimetrically and used to monitor benzene pollution in environmental air samples collected from an oil refinery assessing benzene by different laboratory experimental procedures. The procedures involving whole-cell bioassays determine the concentration of benzene through benzene dioxygenase activity, which allows for direct correlation of oxygen consumption, and through the benzene dihydrodiol dehydrogenase that causes catechol accumulation and restores NADH necessary for the activity of the first enzyme. Oxygen consumption and catechol production deriving from both enzymatic activities are related to benzene concentration and their measurements determined the sensitivity of the system. The results indicated that the sensitivity was enough to detect the benzene vapor at a lower concentration level of 0.01 mM in about 30 min. The possibility for on-line monitoring of benzene concentration by our new recombinant cells results from the fact that no particular treatment of environmental samples is required. This is a major advantage over other biosensors or assays. Moreover, the development of microbial cells that did not require any addition or effectors for the transcription of the specific enzymes, allowed these systems to be more versatile in automated environmental benzene monitoring.

Genetic diversity of bacterial strains isolated from soils, contaminated with polycyclic aromatic hydrocarbons, by 16S rRNA gene sequencing and amplified fragment length polymorphism fingerprinting.

In order to study microbial diversity in a polycyclic aromatic hydrocarbon-impacted soil, 14 bacterial strains were analyzed by 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. Bacterial strains isolated from two different hydrocarbon-polluted sites were identified to the species level by 16S rRNA full-gene sequencing using MicroSeq 16S rRNA gene sequencing. Their genome was subsequently analyzed by high-resolution genotyping with AFLP analysis, in order to monitor species variability and to differentiate closely related strains. Cluster analysis based on AFLP fingerprinting showed intra-specific polymorphism, even among strains with 100% 16S rRNA gene sequence identity. The results show that AFLP is a powerful, highly reproducible and discriminatory tool for revealing genetic relationships in bacterial populations. The ability to differentiate and track related closely microbes is fundamental for studying structure and dynamics of microbial communities in contaminated ecosystems.

[The use of genetically modified microorganisms in agriculture to reduce or eliminate pesticide exposure]. / Utilizzo di microrganismi geneticamente modificati in agricoltura al fine di ridurre o eliminare l'esposizione a pesticidi.

P. graminea is the casual agent of barley leaf stripe. An early selection method of resistant types of barley leaf stripe was realized and validated in this research. This new method, based on Microrganism Genetically Modified (MOGM) GUS2 construction, obtains results comparable with those of classical method, reduces the work time, the use of chemicals (pesticides) and productive plants as greenhouses. Moreover, the use of MOGM GUS2 is restricted in laboratory ambient, therefore the risk of environmental spread is reduced. The early selection method has allowed to estimate the reaction to P. graminea agent in 12 several barley types usually farmed in Italy. The results were compared both, with the classical method data based on artificial clone Dg2 inoculum, and with natural inoculum data obtained in field. At all times we observed a ranking likeness.

[Prevention, safety and prophylactic measures in occupational HIV infection in Italy, the European Union and the USA]. / Misure di prevenzione, sicurezza e profilassi nell'infezione occupazionale da HIV in Italia, Unione Europea e USA.

Numerous guidelines have been issued by Public Health institutions and related authorities in the last few years for the prevention of HIV infection among occupationally exposed workers. Our study was aimed at comparing the regulations and guidelines on this topic that have recently been adopted by Western countries, also taking into account the impact of the problem in current scientific literature. Health-care workers are the category with the highest risk for occupational exposure to HIV principally associated with accidental needlesticks, skin lesions and percutaneous injuries. In preventive and occupational medicine, Italy, the European Union and the USA have founded their recommendations on universal and specific precautions issued by the Center for Disease Control. Moreover, as long ago as 1990 the Italian Ministry of Health issued official guidelines for the prevention of occupational exposure to bloodborne pathogens. Post-exposure management is crucial for the protection of workers at risk. As a consequence of the failure of some monotherapeutic zidovudine treatments, different countries revised their guidelines and recommended the use of a combination of chemotherapeutic drugs for the post-exposure regimen. However, most of the currently available data are derived from efficacy studies of combined therapy on HIV-infected patients. Therefore, further experimental investigations are needed aimed at evaluating the short- and long-term effects of these treatments in the post-exposure protection of workers at risk for HIV infection.

A polymorphism in the 5' region of coagulation factor VII gene (F7) caused by an inserted decanucleotide.

We describe a polymorphism in the 5' region of the coagulation factor VII (FVII) gene, originating from a decanucleotide (CCTATATCCT) insert present in the less frequent allele. This marker can be detected by restriction analysis of polymerase chain reaction products.

Haemophilia in Somalia.

As first step for the development of a programme of haemophilia care, the disease was diagnosed locally in 5 bleeders. These patients were all affected by severe haemophilia A. They were treated with fresh frozen plasma for bleedings. All of them were negative for antibodies to HIV. The potentialities of the Blood Transfusion Centre at Mogadishu are such that with little effort it could produce amounts of cryoprecipitate sufficient to treat most of the patients living in the central region of the country where the capital, Mogadishu, is located.

Induction of relA(p65) and I kappa B alpha subunit expression during differentiation of human peripheral blood monocytes to macrophages.

We evaluated the expression and DNA binding activity of nuclear factor (NF)-kappa B subunits in human peripheral blood monocytes and in monocyte-derived macrophages (MDMs). Constitutive DNA binding activity consisting of p50 homodimers was detected in nuclear extracts from both cell types. An additional complex composed of p50/RelA(p65) heterodimers appeared only in nuclear extracts from 7-day MDMs. Immunoblot analysis showed that the p50 subunit was constitutively expressed in monocytes and MDMs. In contrast, the RelA(p65) subunit was barely detectable in monocytes, but its level increased markedly in MDMs. Analysis of RelA(p65) mRNA revealed that the stability of RelA(p65) mRNA was significantly higher in MDMs, compared with monocytes. In MDMs, an upregulation of I kappa B alpha synthesis as well as the appearance of a novel M(r) 40,000 form of I kappa B alpha were also observed. These results suggest that macrophage differentiation results in the expression of active p50/RelA(p65) heterodimers with the capacity to activate target gene expression. The parallel induction of I kappa B alpha synthesis may allow for the continuous presence of a cytoplasmic reservoir of p50/RelA(p65) complexes that are readily available for inducer-mediated stimulation.

Factor VII gene polymorphisms contribute about one third of the factor VII level variation in plasma.

To assess the role of genetic variation in determining factor VII (FVII) activity and antigen levels we studied a polymorphism located in the 5' region of the gene (5'F7), an intronic mutation (IVS7), and the 353Arg-Gln polymorphism. All the polymorphisms, which showed strong allelic association, analyzed separately or in combination by the one-way analysis of variance, were associated with significantly different FVII levels. The 5'F7 and 353Arg-Gln polymorphic systems, which have very similar allele frequencies, contributed to a similar extent to the total phenotypic variance, whereas the contribution of the IVS7 polymorphism was lower. Genetic variation at the FVII locus, evaluated on combined genotypes, accounted for up to 40% of the phenotype FVII variance. As also shown by the two-way analysis of variance, the use of two out of three markers is advisable, and since the 5'F7 polymorphism can be screened by a simple immunoassay, it should be preferred for population-based studies. No substantial differences between FVII activity and FVII antigen levels were found, thus suggesting that the variation was due to biosynthesis- or stability-mediated mechanisms. The genetic control of FVII levels described in this study plays an important role in determining plasma FVII level variability, which may influence the hemostatic balance.

Molecular analysis of factor VII deficiency in Italy: a frequent mutation (FVII Lazio) in a repeated intronic region.

Molecular defects and polymorphic haplotypes of coagulation factor VII gene were studied in eight unrelated Italian subjects with factor VII deficiency, seven having the factor VII- variant, one the factor VIIR variant. An intron 7 mutation, which alters the consensus donor splice site sequence, was found in six subjects. The presence of the founder effect is suggested by their common geographical origin (a mountain area in the Lazio region) and by the identical polymorphic haplotype underlying the mutation. A different mutation, also located in the 5' monomer of the repeated intron 7 sequence, was found in the heterozygous condition in a subject from Northern Italy. New polymorphic alleles were detected in the repeated intron 7 region in subjects from Eastern Africa. Two missense mutations in codon 97 (Gly-->Cys, Gly-->Ser), the first found in the compound heterozygous condition with the frequent intron 7 mutation, suggest the presence of a hot spot mutation site in the second epidermal growth factor domain. Two neutral dimorphisms at codon 333Ser and 115His were detected, the last in linkage disequilibrium with the 353Arg/Gln polymorphism, and showing differences in frequency in the FVII deficient and control subjects.

Carrier detection and prenatal diagnosis in haemophilia A and B.

Sixty probable carriers of haemophilia from 25 families were studied by using coagulation phenotype and DNA analysis: 33 with haemophilia A and 27 with haemophilia B. Coagulation phenotype was based on factor VIII/IX assay and DNA analysis on the examination of restriction fragment length polymorphisms (RFLPs) within and closely linked to factor VIII or IX: 3 RFLP for factor VIII and 3 for factor IX. The comparison between the coagulation phenotype and RFLP analysis showed the misclassification of 15 females (6 for haemophilia A and 9 for haemophilia B). Four prenatal haemophilia A diagnosis were made by DNA analysis of chorionic villi, taken with a transcervical trophoblastic biopsy, between the 18th and the 11th week.

Carrier detection for hemophilia B: evaluation of multiple polymorphic sites.

DNA analysis was performed in families with hemophilia B. Restriction fragment length polymorphisms (RFLPs) produced by endonucleases Taql, Xmnl, and Ddel were studied by two factor IX genomic probes, F9(VIII) and F9(XIII). Fifty-seven subjects from ten families were investigated; of them, 31 were carriers (11 obligate and 20 potential). Of the potential carriers, ten displayed laboratory features allowing for a phenotypic diagnosis of heterozygosity. Segregation analysis of the markers was informative in 19/20 potential carriers, which belong to nine of the ten studied families. Among the potential carriers, Taql allowed the carriership assessment in 15 (78.9%), Xmnl in 15 (94.7%), and Ddel in two (10.4%). Diagnosis was not possible in one family since a homozygosity in the key individuals with all the employed enzymes (Taql, Xmnl, Ddel, + BamHI) was found. Hemophilia B syndrome in two families likely results from a new mutation. In one family, a first-trimester prenatal diagnosis was performed. The use of RFLP analysis allowed us to improve genetic counseling as compared with the phenotypic evaluation by clotting factor assays. Indeed, evaluation of RFLP increased by 26% the carriership assessment of the potential carriers of the hemophilia B trait.