Physical map of the D6S149-D6S193 region on chromosome 6Q27 and its involvement in benign surface epithelial ovarian tumours

A detailed long range restriction map of the region defined by markers D6S149 and D6S193 on chromosome 6q27 has been constructed. This was achieved by YAC cloning and contig assembling of the same region. Seven YAC clones were found to span the almost 1000 Kb region flanked by the two markers which on the genetic map resulted to be 1.9 cM apart. With some of the characterized YAC clones we undertook a molecular cytogenetic analysis of 20 benign ovarian tumors. The rationale for this was the recent mapping to a region of chromosome 6q27, flanked by markers D6281 and D6S133, of a locus for the SV40-mediated immortalization of human cells (SEN6 gene). Noteworthy we found that the the D6S149-D6S193 region (comprised in the larger D6S281-D6S133 physical interval) was altered in all samples analysed adding support to the occurrence of a immortalization step in this type of tumors.     

Enzyme immobilization on polyglycidylmethacrylate graft-copolymer of different polysaccharides

Comparative results obtained in preparing and characterizing samples of enzymes immobilized by reaction with polyglycidylmethacrylate (PGMA) copolymers with different polysaccharide matrices are reported. Sepharose copolymers having between 25 and 50% synthetic polymer were used to find the best immobilization conditions of horseradish peroxidase (HRP) and glucose-oxidase (GOD) (pH, time, temperature, enzyme cncentration). Activity, enzyme loading and coupling efficiency of immobilized HRP and GOD are greatly dependent on the type of matrix while the polymer content is less important. Coupling efficiencies between 0.8 and 1.5% have been obtained for HRP samples, whereas for GOD samples coupling efficiencies three times greater were obtained. HRP and GOD immobilized samples show K_m′ values greater than those of corresponding free enzymes and this indicates diffusion limitation phenomena. Storage, thermal and operational stability were also studied. In general the storage stability could be considered satisfactory (50% residual activity after 360 days). Sepharose and starch HRP-copolymers had an improved thermal stability compared with that of free enzyme. Residual activity found in continuous operation tests carried out on HRP-immobilized samples turned out to be dependent on support. HRP-PGMA-Cellulose sample gave the best results (50% residual activity after 16 days). PGMA-graft-copolymers have also been used to immobilize other enzymes such as α-amylase, α-chymotrypsin and cellulase.     

TeO2 bolometers to search for Double Beta Decay of130Te: status of art

A 334g TeO₂ crystal is operating since January '93 at Gran Sasso National Laboratories as a thermal detector to search for neutrinoless decay of¹³⁰Te. The reduction of environmental radioactivity is accomplished by means of a 10 cm thick external lead shielding and a plexiglas box against²²²Rn, while an inner ultra-low activity lead shield of at least 3.5 cm in every direction suppresses the residual local radioactivity near the crystal. The good energy resolution and the peak-to-Compton efficiency of the detector allow to understand the main features of the residual background. In particular, we are able to reach a sensitivity in the internal contamination of²³⁸U and²³²Th of the crystal of the order of 10^–13 g/g, better than that of modern mass spectroscopy methods. Pulse acquisition and analysis techniques are reported and discussed.Present limit on neutrinoless decay half-life time of¹³⁰Te obtained with this detector is of the order of 10²²y at 68% C.L., exceeding by one order of magnitude the value quoted by the inclusive geochemical measurements, which have therefore to be attributed mainly to the 2-neutrino channel.On leave from the Dept. of Physics, University of Zaragoza     

A New Technique for the Identification of Surface Background: The Surface Sensitive Bolometers

The main problem for experiments using the bolometric technique to search for rare events is the contribution of surface contamination to the background. In this paper a new technique for the identification of the origin of events will be described. The idea is to shield the main bolometer with bolometric shields. Tests on small and large prototypes and the promising results will be reported.      

A 100-MHz CMOS DAC for video-graphic systems

A 6-b weighted-current-sink video digital-to-analog converter (DAC) with 10-90% rise/fall time of 4 ns, integrated with a double-metal 3-μm CMOS technology, is described. Current-source matching, glitch reduction, and differential switch driving aspects are considered. A circuit solution and a nonconventional layout technique yield a high conversion rate with a standard CMOS technology. Experimental results show that a conversion rate of 100 MHz is achievable. The power consumption is 150 mW and the active chip area is 0.5×1.0 mm² . The differential of 0.1 LSB demonstrates that 8 b of accuracy can be achieved. The integral linearity is 0.5 LSB     

Pathogen detection in milk samples by ligation detection reaction-mediated universal array method

This paper describes a new DNA chip, based on the use of a ligation detection reaction coupled to a universal array, developed to detect and analyze, directly from milk samples, microbial pathogens known to cause bovine, ovine, and caprine mastitis or to be responsible for foodborne intoxication or infection, or both. Probes were designed for the identification of 15 different bacterial groups: Staphylococcus aureus, Streptococcus agalactiae, nonaureus staphylococci, Streptococcus bovis, Streptococcus equi, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus parauberis, Streptococcus uberis, Streptococcus pyogenes, Mycoplasma spp., Salmonella spp., Bacillus spp., Campylobacter spp., and Escherichia coli and related species. These groups were identified based on the 16S rRNA gene. For microarray validation, 22 strains from the American Type Culture Collection or other culture collections and 50 milk samples were tested. The results demonstrated high specificity, with sensitivity as low as 6 fmol. Moreover, the ligation detection reaction-universal array assay allowed for the identification of Mycoplasma spp. in a few hours, avoiding the long incubation times of traditional microbiological identification methods. The universal array described here is a versatile tool able to identify milk pathogens efficiently and rapidly.     

Large mass bolometric detectors for double-beta decay experiments

The authors have developed bolometric detectors with masses of 5.5 g, 20.9 g, and 34 g using TeO₂ crystals for the investigation of ¹³⁰Te, and are developing CaF₂ detectors for double-beta decay search in ⁴⁸Ca, using the luminescence properties of the crystal to improve background rejection. Scintillation of a CaF₂ crystal with 0.03% of Eu doping was observed at 20 mK. A 2.1-g, 0.01%-Eu-doped crystal was used as a bolometer, and a resolution of 50 keV for the double escape peak of ²³²Th was obtained at 60 mK, showing a negligible contribution of the Eu atoms to the crystal heat capacity. Simultaneous detection of light and phonons will allow an efficient background rejection of the ²³⁸U to ²³⁴Th α decay from the ²³⁸U natural series occurring at almost the same energy of the ⁴⁸Ca transition. Background rejection is necessary as even uranium contamination as low as 10^m-10 g/g would make the search impossible     

Occurrence of methicillin-resistant Staphylococcus aureus in dairy cattle herds,related swine farms,and humans in contact with herds

In this study we investigated the circulation of methicillin-resistant Staphylococcus aureus (MRSA) in 2 dairy cattle farms (farm A and B), previously identified as MRSA-positive in bulk tank milk samples, and epidemiologically related to swine farms. Collected specimens included quarter milk samples and nasal swabs from dairy cows, pig nasal swabs collected at both the farm and slaughterhouse level, environmental dust samples, and human nasal swabs from the farms’ owners and workers. The prevalence of MRSA was estimated at the herd level by testing quarter milk samples. The prevalence of MRSA was 4.8% (3/63; 95% confidence interval = 0–10.2%) and 60% (33/55; 95% confidence interval = 47.05–72.95) in farm A and B, respectively. In farm A, MRSA was also isolated from humans, pigs sampled at both farm and slaughterhouse level, and from environmental samples collected at the pig facilities. The dairy cattle facilities of farm A tested negative for MRSA. In farm B, MRSA was isolated from environmental dust samples in both the cattle and pig facilities, whereas nasal swabs collected from cows and from humans tested negative. Sixty-three selected MRSA isolates obtained from different sources in farm A and B were genetically characterized by multilocus sequence typing, spa-typing, ribosomal spacer-PCR, and also tested for the presence of specific virulence genes and for their phenotypical antimicrobial susceptibility by broth microdilution method. Different clonal complex (CC) and spa-types were identified, including CC398, CC97, and CC1, CC already reported in livestock animals in Italy. The MRSA isolates from quarter milk of farm A and B mostly belonged to CC97 and CC398, respectively. Both lineages were also identified in humans in farm A. The CC97 and CC398 quarter milk isolates were also identified as genotype GTBE and GTAF by ribosomal spacer-PCR respectively, belonging to distinct clusters with specific virulence and resistance patterns. The GTBE and GTAF clusters also included swine, environmental, and human isolates from both farms. A high heterogeneity in the genetic and phenotypic profiles was observed in environmental isolates, in particular from farm B. These results demonstrate the possibility of a dynamic sharing and exchange of MRSA lineages or genotypes between different species and farm compartments in mixed-species farms. The risk of transmission between swine and related dairy cattle herds should be considered. Our findings also confirm the zoonotic potential of livestock-associated MRSA and underline the importance of applying biosecurity measures and good hygiene practices to prevent MRSA spread at the farm level and throughout the food production chain.     

Technical note: Identification of Prototheca species from bovine milk samples by PCR-single strand conformation polymorphism

We report the development of a PCR-single strand conformation polymorphism (SSCP) method to identify Prototheca spp. responsible for bovine mastitis: P. zopfii and P. blaschkeae. The method was set up using reference strains belonging to P. zopfii genotype 1, P. zopfii genotype 2, and P. blaschkeae as target species and P. stagnora, and P. ulmea as negative controls. The assay was applied on 50 isolates of Prototheca spp. isolated from bovine mastitic milk or bulk-tank milk samples, and all isolates were identified as P. zopfii genotype 2. We conclude that the described PCR-SSCP approach is accurate, inexpensive, and highly suitable for the identification of P. zopfii genotype 2 on field isolates but also directly on milk, if preceded by a specific DNA extraction method.