Environmental Vibrio parahaemolyticus DNA signatures validation.

Insignia is a novel DNA computational system which uses highly efficient algorithms to compare bacterial genomes and to identify specific DNA signatures to distinguish a target bacterium, or group of bacteria, from all other known bacterial species. It is currently being validated using different bacterial groups, including Vibrio spp. In this study, the genomic analysis by Insignia was conducted on Vibrio parahaemolyticus, a halophilic gram-negative bacteria which constitutes a leading cause of seafood-borne disease. Insignia was used to identify 37 V. parahaemolyticus-specific signatures and to design PCR assays to validate the representative signature sequences by TaqMan essays. The 37 assays targeted loci distributed around the genome and detected genes coding for hypothetical proteins and for proteins involved in adhesion, starvation and virulence. A panel of V. parahaemolyticus environmental strains isolated from the North Adriatic Sea (Italy) and from the Black Sea (Georgia) was used to validate the selected signatures. The signature assays revealed both sensitive and specific and the method allowed a more accurate identification of the tested bacterial strains at the species level when compared to biochemical and PCR standard methods. Using Insignia, it was possible to distinguish two different groups among the strains previously identified as V. parahaemolyticus: most of the strains were included in a "V. parahaemolyticus-like group" showing nearly all of the signatures assayed while a small group of 10 strains contained only a few of the signatures tested. By sequencing the 16S rDNA of this latter group, it was confirmed that they were not V. parahaemolyticus but in fact belonged to other Vibrio species. No significant genome-wide differences were detected between the strains isolated in Italy and in Georgia though the very different geographical origin.

465 nm laser sources by intracavity frequency doubling using a 49-edge-emitters laser bar.

A compact blue laser was generated by intracavity frequency doubling based on quasi-phase-matched second-harmonic generation (SHG) in a MgO-doped periodically poled lithium niobate bulk crystal. A 49 single-transverse-mode edge-emitters laser bar with antireflective coating was used as a pump source. An optical output power of 1.2 W SHG of blue lights at 465 nm is generated at 45 A injection current, equivalent to an overall wall-plug efficiency of 1.33%.

Do users of the intrauterine system (Mirena) have different genital symptoms and vaginal flora than users of the intrauterine contraceptive device?

The copper intrauterine contraceptive device (IUCD) is strongly associated with bacterial vaginosis (BV). Hormonal influences may play a role in the control of vaginal flora. It is unclear whether use of the progesterone-incorporated intrauterine system (IUS; Mirena) is associated with abnormal vaginal flora or genital symptoms. One hundred and seventy-two women were assessed for symptoms and abnormal vaginal flora prior to and at intervals after insertion of either a copper IUCD or an IUS. Women were significantly more likely to have developed an abnormal vaginal discharge 4-6 weeks after insertion of an IUCD compared with an IUS (27% cf. 14%, P = 0.04), although this trend was not significant six months postinsertion. More women with an IUCD developed BV compared with an IUS at 4-6 weeks and six months. However, there were insufficient numbers of women with BV to demonstrate any significant difference between the vaginal flora of the two groups.

Beverage caffeine intake in US consumers and subpopulations of interest: estimates from the Share of Intake Panel survey.

Concerns exist about the potential adverse health effects of high consumption of dietary caffeine, especially in children and pregnant women. Recommended caffeine intakes corresponding to no adverse health effects have been suggested recently for healthy adults (400-450 mg/day), for women contemplating pregnancy (300 mg/day), and for young children age 4-6 years (45 mg/day). To determine whether current caffeine intake approaches these levels, intake from major dietary sources (coffee, tea and carbonated soft drinks) were measured in 10,712 caffeinated beverage consumers in the 1999 US Share of Intake Panel, a targeted beverage survey. Mean caffeine intakes in adult caffeinated beverage consumers ranged from 106 to 170 mg/day (90th percentile intake was 227-382 mg/day). In children 1-5 and 6-9 years, mean caffeine intakes were 14 and 22 mg/day, respectively; corresponding 90th percentile intakes were 37 and 45 mg/day. Pregnant women consumed an average of 58 mg/day (157 mg/day at the 90th percentile), and women of reproductive age ingested 91-109 mg/day (229-247 mg/day at the 90th percentile). These data show that while mean caffeine intakes are within recommended safe levels, heavy consumers of certain subpopulations, including young children and women contemplating pregnancy, might benefit from dietary advice.

Detection of Bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost.

Control of Bordetella pertussis in the community is hampered by slow and insensitive diagnostic tests. We therefore examined the accuracy and cost of culture, direct fluorescent antibody (DFA) staining, and PCR in a routine clinical laboratory. Six hundred thirty seven nasopharyngeal swabs and aspirates in casamino acids transport medium were cultured, stained with polyclonal (Difco), and monoclonal (BL-5 and Accu-Mab) anti-B. pertussis reagents, and amplified by an IS481-specific PCR. PCR products were detected by a hybridization-enzyme immunoassay kit (Gen-eti-k DEIA, DiaSorin), with confirmation by a second PCR in a separate laboratory. Sensitivities and specificities of culture, polyclonal DFA, monoclonal DFA, and PCR were 36 and 100%, 11.4 and 94.6%, 8.3 and 98. 4%, and 95.0 and 99.3%, respectively, with a prevalence of 15.7%. The DFA tests were the most economical, and the PCR cost was 31% higher than culture. This study suggests that with minor improvements in economy, pertussis PCR can be implemented in a clinical laboratory with marked improvement in diagnostic accuracy.

Temperature-induced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation?

Vibrio cholerae cells were incubated at 4 degrees C in nutrient-limited artificial seawater (ASW) microcosms. Plate counts declined from 8 x 10(5) to less than 2 c.f.u. ml-1 in about 23 d. When samples of microcosms were shifted to 30 degrees C, plate counts increased to 2.2 x 10(5) c.f.u. ml-1 in 72 h. An experiment was performed to determine whether culturable cells obtained after temperature upshifts were the result of 'resuscitation', or outgrowth, of nonculturable cells or of cell division and growth of the few culturable cells that remained in samples. Prior to temperature upshift, samples from the microcosms were diluted 10- and 100-fold in filter-sterilized (0.1 microns) ASW from the microcosms. Undiluted, 1/10, and 1/100 diluted samples recovered culturability to about 2.2 x 10(5) c.f.u. ml-1 within 72 h of temperature upshift. If resuscitation of nonculturable cells had occurred, the resultant number of culturable cells in diluted samples would have been 1/10 and 1/100 that of undiluted samples, respectively. In microcosms where plate counts had declined to less than 1 c.f.u. ml-1, 1/100 diluted samples did not regain culturability, i.e. no culturable cells remained from which growth could occur. Our conclusions are that in the experiments reported here, recovery of culturable cells on temperature upshifts resulted from growth and that there were no growth-inhibiting factors in the spent growth medium, supported by the finding that about 10(2) recovered V. cholerae cells ml-1 inoculated into filter-sterilized microcosm ASW grew to about 6.2 x 10(5) c.f.u. ml-1 in 24 h, confirming that V. cholerae is capable of significant growth in ASW.

The development and applications of sucralose, a new high-intensity sweetener.

Sucralose is a new sweetener discovered during a collaborative research program between Tate & Lyle and Queen Elizabeth College of the University of London. It is made by selective substitution of sucrose hydroxyl groups by chlorine, resulting in a highly intense (600x) sugarlike sweetness and exceptional stability at both high temperature and low pH. The research leading to the discovery of sweetness in differently halogenated sucrose is described, as well as the development of sucralose and the process of safety testing and government approval. Finally, sucralose properties and applications in Canada's food and beverage industries are discussed.

Benthic Distribution of Sewage Sludge Indicated by Clostridium perfringens at a Deep-Ocean Dump Site.

Clostridium perfringens in sediment samples collected at the Deep Water Municipal Sewage Sludge Disposal Site (also called the 106-Mile Site), off the coast of New Jersey, was enumerated. The counts of C. perfringens found in sediment samples collected within and to the southwest of the 106-Mile Site were significantly elevated (P < 0.01) compared with counts of samples from reference stations of similar depth (2,400 to 2,700 m), topography, and distance from the continental shelf, indicating that the benthic environment was contaminated by sewage dumping at this site. Low counts of C. perfringens in sediment samples collected at stations between the base of the continental shelf and the 106-Mile Site indicated that coastal runoff was not a significant source of contamination. Elevated counts were observed for samples up to 92 km to the southwest, whereas low counts were obtained for samples from stations to the east of the 106-Mile Site. This distribution is consistent with previous model predictions of sludge deposition. In areas heavily impacted by sludge dumping, C. perfringens counts were generally highest in the top 1 cm of sediment and exceeded 9,000 CFU g (dry weight) of sediment. The patterns of C. perfringens dispersal observed in this study have proved useful for selection of heavily impacted areas and control stations for further ecological evaluation by a multidisciplinary research team.

Evaluation of radiolabeled and colorimetric DNA probes in comparison with an antigen screening assay for the detection of Salmonella from poultry farms.

A total of 48 environmental drag-swab samples from various poultry farms were tested for the presence of Salmonella spp. by culture, an enzyme-linked immunosorbent assay-based Salmonella antigen screening (SAS) assay, and two DNA probes (radiolabeled and colorimetric). The radiolabeled DNA probe was allowed to hybridize with culture-positive samples (n = 8) and was found to detect Salmonella spp. in all cases (100%). Both of the probes, subsequently hybridized with culture-negative samples (n = 8), were observed to yield good agreement (91%) with the culture findings. The remaining samples (n = 32) were tested by the SAS assay, and where there was no agreement between the culture and SAS, samples were further examined by the DNA probes. Results using both probes agreed with those obtained by culturing the samples but did not agree with the SAS assay result when the ratio of samples tested to samples positive (S/P) cutoff value used was 0.5.

Antibiotic resistance indexing of Escherichia coli to identify sources of fecal contamination in water.

A total of 202 Escherichia coli isolated from urban and rural water were tested with 11 antibiotics to assess the prevalence of antibiotic resistance from each source. Urban waters harbored higher percentages of resistant E. coli strains than rural waters. Antibiotic-resistant E. coli may offer an index of water quality related to source.

Direct detection of Salmonella spp. in estuaries by using a DNA probe.

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.

Mixed lymphocyte cultures using automated blood cell counters.

We compared the standard mixed lymphocyte culture technique with a new technique based on cell count and size distribution in a series of histocompatibility tests on 24 patients and 39 potential donors for bone marrow transplantation. We found that suspensions in which lymphocytes were undergoing transformation could be distinguished from those in which they were not after a 10-day incubation period by measuring the leucocyte count and size distribution in a Coulter S+ IV automated counter. The leucocyte count was higher and the size distribution was wider in suspensions where the lymphocytes were transforming. The correlation between relative response indices calculated using tritiated thymidine uptake or total nuclear mass was close (r = 0.7; p less than 0.001) although there were major differences in the response as assessed by each of the two techniques in some individual cases. We feel this method is suitable to screen large numbers of unrelated donors.

Simple, rapid method for direct isolation of nucleic acids from aquatic environments.

Direct isolation of nucleic acids from the environment may be useful in several respects, including the estimation of total biomass, detection of specific organisms and genes, estimations of species diversity, and cloning applications. We have developed a method that facilitates the concentration of microorganisms from aquatic samples and the extraction of their nucleic acids. Natural water samples of 350 to greater than 1,000 ml are concentrated on a single cylindrical filter membrane (type SVGS01015; Millipore Corp., Bedford, Mass.), and cell lysis and proteolysis are carried out within the filter housing. Crude, high-molecular-weight nucleic acid solutions are then drawn off the filter. These solutions can be immediately analyzed, concentrated, or purified, depending on the intended application. The method is simple, rapid, and economical and provides high-molecular-weight chromosomal DNA, plasmid DNA, and speciated RNAs which comigrate with 5S, 16S, and 23S rRNAs. The methods presented here should prove useful in studying both the ecology and the phylogeny of microbes that resist classical culture methods.

Paradigms and crises in psychoanalysis.

The use of T. S. Kuhn's Structure of Scientific Revolutions as a model for the history of psychoanalysis is addressed. It is shown that his categories have been regularly misapplied in the interest of establishing the scientific status of psychoanalysis, or of proposing a new theoretical structure. It is argued that Kuhn's model may nevertheless be useful for the history of psychoanalysis on condition that its categories are used precisely, and their correspondence to the data is tested rather than assumed.

Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.

Relation of free silicone to human breast carcinoma.

Of 12 women with carcinoma of the breast and coexistent silicone mastopathy, nine had had injections of liquid silicone for breast augmentation; three had leaking silicone-gel prostheses. The clinical findings indicated that early diagnosis was obscured by the silicone-induced mastopathy, which rendered the interpretation of physical findings and mammograms difficult. The pathologic findings were suggestive of a possible adverse effect of the presence of free silicone within the breast tissue, axillary nodes, and axillary fat. Although no causal relationship between silicone and breast carcinoma is implied, a heightened awareness of the possible coexistence of silicone mastopathy and breast carcinoma is necessary.