Development of a novel method for detection of Clostridium difficile using HS-SPME-GC-MS.

AIMS: A novel method has been developed that allows successful differentiation between Clostridium difficile culture-positive and culture-negative stool samples based on volatile organic compound (VOC) evolution and detection by headspace solid-phase microextraction coupled with gas chromatography mass spectrometry (HS-SPME-GC-MS). METHODS AND RESULTS: The method is based on the activation of p-hydroxyphenylacetate decarboxylase produced by Cl. difficile and the detection of a specific VOC, that is 2-fluoro-4-methylphenol from an enzyme substrate. In addition, other VOCs were good indicators for Cl. difficile, that is isocaproic acid and p-cresol, although they could not be used alone for identification purposes. One hundred stool samples were tested, of which 77 were positive by culture. Detection using HS-SPME-GC-MS allowed confirmation of the presence of Cl. difficile within 18 h with a sensitivity and specificity of 83·1 and 100%, respectively. CONCLUSIONS: It is recommended that this new approach could be used alongside conventional methods for Cl. difficile detection, including toxin detection methods, which would allow any false-negative results to be eliminated. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify Cl. difficile-positive stool samples by the analysis of VOCs could allow the development of a VOC detection device which could allow rapid diagnosis of disease and hence prompt treatment with appropriate antibiotics.

Comparison of two selective media for the recovery of Clostridium difficile from environmental surfaces.

Two culture media were compared for their ability to isolate Clostridium difficile from environmental sites within a UK hospital. The media were cefoxitin-cycloserine-egg yolk agar plus lysozyme (CCEY/L) and chromID C. difficile. A wide range of environmental surfaces was sampled using sterile sponges (Polywipes) and these were inoculated on to both media. C. difficile was recovered from 105 of 496 sites (21%) using a combination of both media. The sensitivity of chromID C. difficile was 87.6% compared with 26.6% for CCEY/L (P < 0.0001). chromID C. difficile performed significantly better than CCEY/L for the recovery of C. difficile from the environment.

Long-term results of screening with magnetic resonance imaging in women with BRCA mutations.

BACKGROUND: The addition of breast magnetic resonance imaging (MRI) to screening mammography for women with BRCA mutations significantly increases sensitivity, but there is little data on clinical outcomes. We report screening performance, cancer stage, distant recurrence rate, and breast cancer-specific mortality in our screening study. METHODS: From 1997 to 2009, 496 women aged 25 to 65 years with a known BRCA1/2 mutation, of whom 380 had no previous cancer history, were enrolled in a prospective screening trial that included annual MRI and mammography. RESULTS: In 1847 screening rounds, 57 cancers were identified (53 screen-detected, 1 interval, and 3 incidental at prophylactic mastectomy), of which 37 (65%) were invasive. Sensitivity of MRI vs mammography was 86% vs 19% over the entire study period (P<0.0001), but was 74% vs 35% from 1997 to 2002 (P=0.02) and 94% vs 9% from 2003 to 2009 (P<0.0001), respectively. The relative sensitivities of MRI and mammography did not differ by mutation, age, or invasive vs non-invasive disease. Of the incident cancers, 97% were Stage 0 or 1. Of 28 previously unaffected women diagnosed with invasive cancer, 1 BRCA1 mutation carrier died following relapse of a 3 cm, node-positive breast cancer diagnosed on her first screen at age 48 (annual breast cancer mortality rate=0.5%). Three patients died of other causes. None of the 24 survivors has had a distant recurrence at a median follow-up of 8.4 years since diagnosis. CONCLUSION: Magnetic resonance imaging surveillance of women with BRCA1/2 mutations will detect the majority of breast cancers at a very early stage. The absence of distant recurrences of incident cancers to date is encouraging. However, longer follow-up is needed to confirm the safety of breast surveillance.

Risk factors for inhibitor formation in haemophilia: a prevalent case-control study.

Inhibitor formation is a major complication of haemophilia treatment. In a prevalent case-control study, we evaluated blood product exposure, genotype and HLA type on haemophilia A inhibitor formation. Product exposure was extracted from medical records. Genotype was determined on stored DNA samples by detection of virtually all mutations-SSCP (DOVAM-S) and subcycling PCR. HLA typing was performed by PCR amplification and exonuclease-released fluorescence. Cases experienced higher intensity factor, 455 vs. 200 U per exposure, P < 0.005, more frequent central nervous system (CNS) bleeding, seven of 20 (35.0%) vs. one of 57 (1.7%), P = 0.001 and more commonly from inhibitor families, seven of 20 (35.0%) vs. zero of 57 (0%), P < 0.001, and African-American, 12 of 63 (19.0%) vs. six of 117 (5.1%), P = 0.015. Among the latter, CNS bleeding was more commonly the initial bleed, 60% vs. 0%, P < 0.001, and survival was shorter, 14 vs. 38 yr, P = 0.025. Inhibitor formation was uncommon in those with missense mutations, two of 65 (3.1%) vs. 31 of 119 (26.0%), P = 0.008, and unrelated to factor VIII immunogenic epitope, P = 0.388, or HLA type, P > 0.100. Genotype was not associated with race. Time to immune tolerance was shorter for titres <120 vs. > or = 120 BU/mL, six vs. 16 months, P < 0.01, but unaffected by tolerizing dose regimen, P > 0.50. Inhibitor formation is associated with high intensity product exposure, CNS bleeding, African-American race and low frequency of missense mutations. The ideal time to initiate prophylaxis to reduce CNS bleeding and inhibitor formation will require prospective studies.

Clinical development of leukocyte cyclooxygenase 2 activity as a systemic biomarker for cancer chemopreventive agents.

Advancement of cancer prevention and therapy requires clinical development of systemic biomarkers of pharmacological efficacy of the agent under scrutiny. Curcumin, a polyphenol derived from Curcuma spp., has shown wide-ranging chemopreventive activity in preclinical carcinogenic models, in which it inhibits cyclooxygenase (COX)-2 at the transcriptional level. COX-2 has been implicated in the development of many human cancers. To explore the inhibition of COX-2 activity as a systemic biomarker of drug efficacy, a biomarker of potential use in clinical trials of many chemopreventive drugs known to inhibit this enzyme, we measured COX-2 protein induction and prostaglandin E(2) (PGE(2)) production in human blood after incubation with lipopolysaccharide (LPS). When 1 microM curcumin was added in vitro to blood from healthy volunteers, LPS-induced COX-2 protein levels and concomitant PGE(2) production were reduced by 24% and 41%, respectively (P < 0.05 by ANOVA). To test whether effects on COX-2 activity could also be measured after oral dosing in humans, we conducted a dose-escalation pilot study of a standardized formulation of Curcuma extract in 15 patients with advanced colorectal cancer. Basal and LPS-mediated PGE(2) production was measured in blood, twice pretreatment and on days 1, 2, 8, and 29 of treatment. Analysis of basal and LPS-induced PGE(2) production during treatment demonstrated a trend toward dose-dependent inhibition (P < 0.005 by regression analysis), but there was no significant difference compared with values from pretreatment time points. Measurement of leukocyte COX-2 activity should be considered in clinical trials of other agents likely to inhibit this isozyme.

Human germline mutation in the factor IX gene.

The molecular epidemiology of factor IX germline mutations in patients with hemophilia B has been studied in detail because it is an advantageous model for analyzing recent germline mutations in humans. It is estimated that mutations have been defined in the majority of nucleotides that are the target for mutation. The likelihood that a factor IX missense mutation will cause disease correlates with the degree of evolutionary conservation of the amino acid. Mutation rates per base-pair have been estimated after careful consideration and correction for biases, predicting about 76 de novo mutations per generation per individual resulting in 0.3 deleterious changes. The male-to-female sex ratio of mutation varies with the type of mutation. There is evidence for a maternal age effect and an excess of non-CpG G:C to A:T transitions. The factor IX mutation pattern is similar among geographically, racially and ethnically diverse human populations. The data support primarily endogenous mechanisms of germline mutation in the factor IX gene. Mutations at splice junctions are compatible with simple rules for predicting disease causing mutations.

Calcifying fibrous pseudotumor versus inflammatory myofibroblastic tumor: a histological and immunohistochemical comparison.

Calcifying fibrous pseudotumor (CFP), a recently described lesion, is characterized by a predominantly lymphoplasmacytic infiltrate with abundant hyalinized collagen and psammomatous or dystrophic calcifications. The cause and pathogenesis are unclear, but it has been postulated that CFP may represent a sclerosing end stage of inflammatory myofibroblastic tumor (IMT). We compared the histological and immunohistochemical profiles of seven cases diagnosed as CFP and seven as IMT. Histologically, the CFP demonstrated varying degrees of calcifications in addition to fibroblastic proliferation admixed with inflammatory cells composed of lymphocytes, eosinophils, and mast cells. The IMTs rarely contain calcifications and had a myofibroblastic proliferation varying from hyalinized acellular collagen to florid fibroblastic proliferations simulating sarcoma. The inflammatory component was composed primarily of plasma cells and lymphocytes, sometimes arranged as lymphoid aggregates with germinal centers. All CFP cases were diffusely positive for factor XIIIa and negative for smooth muscle actin, muscle-specific actin, and CD34. All IMTs demonstrated diffuse positivity for actin, variable positivity for CD34, and focal positivity for Factor XIIIa. This study demonstrates certain distinct histologic, immunohistochemical, and electron microscopic features between IMTs and CFPs.

Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients with colorectal cancer.

Curcuma spp. extracts, particularly the dietary polyphenol curcumin, prevent colon cancer in rodents. In view of the sparse information on the pharmacodynamics and pharmacokinetics of curcumin in humans, a dose-escalation pilot study of a novel standardized Curcuma extract in proprietary capsule form was performed at doses between 440 and 2200 mg/day, containing 36-180 mg of curcumin. Fifteen patients with advanced colorectal cancer refractory to standard chemotherapies received Curcuma extract daily for up to 4 months. Activity of glutathione S-transferase and levels of a DNA adduct (M(1)G) formed by malondialdehyde, a product of lipid peroxidation and prostaglandin biosynthesis, were measured in patients' blood cells. Oral Curcuma extract was well tolerated, and dose-limiting toxicity was not observed. Neither curcumin nor its metabolites were detected in blood or urine, but curcumin was recovered from feces. Curcumin sulfate was identified in the feces of one patient. Ingestion of 440 mg of Curcuma extract for 29 days was accompanied by a 59% decrease in lymphocytic glutathione S-transferase activity. At higher dose levels, this effect was not observed. Leukocytic M(1)G levels were constant within each patient and unaffected by treatment. Radiologically stable disease was demonstrated in five patients for 2-4 months of treatment. The results suggest that (a) Curcuma extract can be administered safely to patients at doses of up to 2.2 g daily, equivalent to 180 mg of curcumin; (b) curcumin has low oral bioavailability in humans and may undergo intestinal metabolism; and (c) larger clinical trials of Curcuma extract are merited.

Spontaneous microdeletions and microinsertions in a transgenic mouse mutation detection system: analysis of age, tissue, and sequence specificity.

A total of 3497 independent spontaneous mutations were examined using the Big Blue transgenic mouse mutation detection system. Base substitutions predominate, although 16% of somatic and germline mutations are microdeletions, microinsertions, or deletions combined with insertions. The pattern of microdeletions and microinsertions is similar in both the lacI transgene and the human p53 gene. Single-base deletions (D1) and insertions (I1) are evenly distributed in the lacI transgene, whereas microdeletions from 2 to 50 bp are clustered at two regions (bp 129-228 and 529-628). The pattern of microdeletions and microinsertions is similar between young (< or =3 months) and old (25 months) mice. Brain tissue has a paucity of deletions combined with insertions when compared with that of thymus and nine other tissues (P = 0.01). A 16-bp deletion at lacI base position 272 is a tissue-specific hotspot preferentially occurring in brain. Approximately 68 and 93% of D1 and I1, respectively, occur at mononucleotide repeats. The frequencies of D1 and I1 in mononucleotide repeats increase in an exponential manner with the length of the repeat. The lacI transgene shows similarity to the human p53 gene in the pattern of microdeletions and microinsertions and the size distribution of microdeletions.

Frequency of recent retrotransposition events in the human factor IX gene.

Two germline retrotransposition mutations of recent origin were observed in 727 independent mutations (0.28%) in the human factor IX gene (F9) of patients with hemophilia B: 1) a 279 bp insertion in exon H originating from an Alu family of short interspersed elements not previously known to be active and, 2) a 463 bp insertion in exon E of a LINE1 element originating in the maternal grandmother. If the rates of recent germline mutation in F9 are typical of the genome, a retrotransposition event is estimated to occur somewhere in the genome of about one in every 17 children born. Analysis of other estimates for retrotransposition frequency and overall mutation rates suggests that the actual rate of retrotransposition is likely to be in the range of one in every 2.4 to 28 live births.

Effects of dietary curcumin on glutathione S-transferase and malondialdehyde-DNA adducts in rat liver and colon mucosa: relationship with drug levels.

Curcumin prevents colon cancer in rodent models. It inhibits lipid peroxidation and cyclooxygenase-2 (COX-2) expression and induces glutathione S-transferase (GST) enzymes. We tested the hypothesis that 14 days of dietary curcumin (2%) affects biomarkers relevant to cancer chemoprevention in the rat. Levels of inducible COX-2, as reflected by prostaglandin E(2) production by blood leukocytes, were measured ex vivo. Total GST activity and adducts of malondialdehyde with DNA (M(1)G), which reflect endogenous lipid peroxidation, were measured in colon mucosa, liver, and blood leukocytes. Curcumin and its metabolites were analyzed by high-performance liquid chromatography in plasma, and its pharmacokinetics were compared following a diet containing 2% curcumin versus intragastric (i.g.) administration of curcumin suspended in an amphiphilic solvent. The curcumin diet did not alter any of the markers in the blood but increased hepatic GST by 16% and decreased colon M(1)G levels by 36% when compared with controls. Administration of carbon tetrachloride during the treatment period increased colon M(1)G levels, and this increase was prevented by dietary curcumin. Dietary curcumin yielded low drug levels in the plasma, between 0 and 12 nM, whereas tissue concentrations of curcumin in liver and colon mucosa were 0.1--0.9 nmol/g and 0.2--1.8 micromol/g, respectively. In comparison with dietary administration, suspended curcumin given i.g. resulted in more curcumin in the plasma but much less in the colon mucosa. The results show that curcumin mixed with the diet achieves drug levels in the colon and liver sufficient to explain the pharmacological activities observed and suggest that this mode of administration may be preferable for the chemoprevention of colon cancer.

Mutation frequency is reduced in the cerebellum of Big Blue mice overexpressing a human wild type SOD1 gene.

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by motor neuron degeneration. A similar disease phenotype is observed in mice overexpressing a mutant human hSOD1 gene (G93A, 1Gurd(1)). Mice transgenic for lacI (Big Blue) and human mutant (1Gurd(1), Mut hSOD1) or wild type (2Gur, Wt hSOD1) SOD1 genes were used to examine spontaneous mutation, oxidative DNA damage, and neurodegeneration in vivo. The frequency and pattern of spontaneous mutation were determined for forebrain (90% glia), cerebellum (90% neurons) and thymus from 5-month-old male mice. Mutation frequency is not elevated significantly and mutation pattern is unaltered in Mut hSOD1 mice compared to control mice. Mutation frequency is reduced significantly in the cerebellum of Wt hSOD1 mice (1.6x10(-5); P=0.0093; Fisher's Exact Test) compared to mice without a human transgene (2.7x10(-5)). Mutation pattern is unaltered. This first report of an endogenous factor that can reduce in vivo, the frequency of spontaneous mutation suggests potential strategies for lowering mutagenesis related to aging, neurodegeneration, and carcinogenesis.

Evidence that proximal multiple mutations in Big Blue transgenic mice are dependent events.

To characterize the nature of multiple mutations in the tissues of an intact animal, the Big Blue transgenic mouse mutation detection system was used to examine 1459 mutants from eight normal tissues and 507 mutants from 11 tumors. Multiple mutations occurred and predominantly doublet mutants were identified (i.e. two mutations within one mutant lacI gene), but multiplets of up to five mutations were observed. The frequency of doublets in normal tissues and spontaneous tumors from p53-deficient mice was enhanced to the same degree (660 and 667 fold, respectively) over that expected for two independent mutational events. Doublets, multiplets and singlets have similar patterns of mutation. The distance between mutations in doublets fits an exponential distribution, not that expected for randomly spaced events, suggesting that many doublets occur in rapid succession within the same cell cycle.

High plating density improves Big Blue system efficiency without loss of sensitivity.

To increase efficiency in the Big Blue system, the plating density was increased from 15000 to 30000 or 45000 plaque forming units (pfus) per plate by increasing the density of the E. coli lawn and decreasing individual plaque size. Small plaque size ensured minimal overlap of the plaques. Liver from one 3- and one 25-month-old mouse (low and high mutation frequencies, respectively) was analyzed and neither plating density nor plaque size affected mutant/mutation frequency and pattern. The color intensity of particular mutant plaques was not affected by plaque size or plating density. Optimal sensitivity is achieved by sequencing mutants to calculate the mutation frequency from the mutant frequency and to identify altered patterns of mutation. Detailed effort and cost accounting of the Big Blue system (including mouse handling, DNA extraction, plaque screening, plaque purification, and DNA sequencing) reveals that one-quarter of the total effort is devoted to plating and screening of plates. This effort is reduced two fold with high plating density. The total cost of the Big Blue system is reduced by 17%. The total cost of the High Plating Density Big Blue system is now only 12% more costly than a selectable assay and offers an extensively validated system with a large mutation database representing a decade of effort.

Calcifying fibrous pseudotumor involving the neck of a five-week-old infant. Presence of factor XIIIa in the lesional cells.

Calcifying fibrous pseudotumor is an uncommon lesion characterized by hyalinized collagen, psammomatous or dystrophic calcifications, and a predominantly lymphoplasmacytic infiltrate. Although the pathogenesis is unclear, a possible relationship with other inflammatory "pseudotumors" has been proposed. We describe the pathology of two right neck calcifying fibrous pseudotumors present in a five-week-old female infant. The masses had many of the pathologic features of calcifying fibrous pseudotumor. The presence of a florid, mixed infiltrate, and the occurrence of more than one lesion in the same patient, favor the proposal that calcifying fibrous pseudotumor may be a sclerosing end stage of inflammatory myofibroblastic tumor. However, the presence of a previously undescribed participation of Factor XIIIa-positive cells suggests that the tumor may be of dermal dendrocyte origin.

Spontaneous mutation frequency and pattern in Big Blue mice fed a vitamin E-supplemented diet.

Endogenous oxidative DNA damage caused by normal cellular processes may play a vital role in carcinogenesis. To directly test the hypothesis that antioxidants will protect DNA from oxidative damage in vivo, Big Blue((R)) mice were fed either a control diet (66 IU vitamin E/kg diet) or a high-dose vitamin E diet containing 1000 IU vitamin E/kg diet of racemic d,l-alpha-tocopherol acetate from conception until 3 months of age. Using the standard Big Blue((R)) protocol, 15.5 million plaque forming units (pfu) were examined from five tissues (heart, liver, adipose tissue, thymus, and testis) of three control and three high-dose vitamin E supplemented male mice generating 433 mutants, which represented 373 independent mutations upon sequencing the lacI transgene. The alpha-tocopherol tissue concentration increased with high-dose vitamin E supplementation. In four of the tissues, individually or combined, mutation frequency changed little if any with vitamin E supplementation. In adipose tissue, which accumulated the highest levels of vitamin E, mutation frequency was significantly reduced with high-dose vitamin E supplementation (P = 0.047). Within the constraints of sample size, the pattern of mutation in adipose tissue was not altered significantly (P = 0.40). When data from all tissues were combined, a reduction in G:C --> T:A transversions was observed (P = 0.044). These results may have implications for cancer chemoprevention and provide insight into the efficacy of vitamin E supplementation in reducing spontaneous oxidative DNA damage in vivo. More dramatic alterations of mutation frequency and pattern may be observed with higher doses of vitamin E and substitution of the racemic supplement with d-alpha-tocopherol acetate.

Characterization of zinc-depleted alanyl-tRNA synthetase from Escherichia coli: role of zinc.

To evaluate the role of zinc in Escherichia coli alanyl-tRNA synthetase, hydrodynamic measurements and circular dichroism spectra were obtained for the zinc-depleted protein and compared with those of the native enzyme. At a protein concentration of 5 mg ml(-1), pH 7.5, the sedimentation coefficient (s(20,w)) was 6.3 S and was virtually independent of temperature between 10 and 37 degrees C, similar to the results reported for the native form. However, the s(20,w) now decreased significantly as the concentration increased, indicative of a possible change in conformation. The s(20,w) value did not appear to change as the pH was increased to 9.5. In standard buffer with 3.3 M added urea, a single peak with a s(20,w) of 3.6 S was obtained and with 6.6 M added urea, a peak with a s(20,w) of 2.7 S was seen. Added Gd-HCl (6 M) gave a single peak with s(20,w) of 2. 0 S. Like the native form, laser light scattering studies indicated some heterogeneity and a radius of 6.4 nm which was virtually independent of concentration and temperature in the range of 10-37 degrees C. At 25 degrees C, a diffusion coefficient (D(20,w)) of 3.3 x 10(-7) cm(2) s(-1) was obtained. The combination of s(0)(20,w) and D(20,w) yielded a molecular mass of approximately 179 kDa, which is slightly less than that reported for the native dimeric form (186 kDa). The intrinsic viscosity at 25 degrees C was extrapolated to 5. 3 ml g(-1), a value significantly higher than that reported for the native form, which increased with temperature. These results indicate some conformational and flexibility changes from the native to the zinc-depleted form, which may explain differences in activity. Furthermore, urea denaturation experiments demonstrate the role of zinc in stabilization of AlaRS structure.

Tandem-base mutations occur in mouse liver and adipose tissue preferentially as G:C to T:A transversions and accumulate with age.

Tandem-base mutations (TBM) are associated with ultraviolet light and other mutagens. Herein, we report an age- and tissue-specific difference in the frequency of spontaneous TBM in Big Blue transgenic mice. A total of 390 mutants from liver and adipose tissue contained 17 and 4 TBM, respectively, while no TBM were detected in 683 mutants from six other tissues. There was a proportional increase in the frequency of TBM in liver with age (29 days postconception to 25 months of age). Nine TBM (43%) were GG to TT transversions that preferentially occurred at specific sites. The remaining 12 mutants contained at least one transversion mutation each. We speculate that the increase of TBM in liver and adipose tissue with age is due to chronic mutagen exposure, perhaps derived from fat in the diet.

The Big Blue(R) transgenic mouse mutation detection assay: the mutation pattern of sectored mutant plaques.

There are mutational artifacts in the Big Blue(R) assay and it is important to characterize the source and nature of these mutations. Differences were reported in the mutation patterns of a small sample of 23 sectored and 91 circular mutant plaques derived from skin using the Big Blue(R) transgenic mouse mutation detection system [G. R. Stuart, N.J. Gorelick, J.L. Andrews, J.G. de Boer, B.W. Glickman, The genetic analysis of lacI mutations in sectored plaques from Big Blue transgenic mice, Environ. Mol. Mutagen 28 (1996) 385-392.]. We have extended these observations by analyzing 46 sectored and 224 circular mutant plaques derived from seven tissues. The frequency of sectored mutant plaques is estimated to be 16% with no significant variation with tissue type. However, the patterns of mutation for sectored mutants and mouse-derived mutations differed significantly (p=0.04). Base substitutions in sectored mutant plaques do not show the asymmetries found in circular mutants consistent with integration of a GC rich transgene into the AT rich mammalian genome. Sectored mutants have mutation patterns consistent with a mixture of mouse, in vitro and Escherichia coli-derived mutations. Data on the relative frequencies of different mutant plaque morphologies suggests that overlapped plaques are substantially contaminated by sectored plaques at recommended plating densities.