Mychael Danna: l'atípic

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Unambiguous Detection of Multiple TP53 Gene Mutations in AAN-Associated Urothelial Cancer in Belgium Using Laser Capture Microdissection

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n = 5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5-8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n = 4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n = 7) or transversions (n = 9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time

Des-, syn- and anti-oxyimino-Δ3-cephalosporins. Intrinsic reactivity and reaction with RTEM-2 serine β-lactamase and D-alanyl-D-alanine-cleaving serine and zinc-containing peptidases

The presence and configuration (syn or anti) of an oxyimino group in the 7 (beta)-acyl side chain of 3-cephems do not modify the intrinsic reactivity of the beta-lactam ring, but have highly enzyme-specific effects. When compared with the corresponding desoxyimino beta-lactam compound: (i) with the plasmid-mediated Escherichia coli RTEM-2 serine beta-lactamase, the substrate activity of the anti isomer is increased and that of the syn isomer is decreased; (ii) with the Streptomyces R61 serine D-alanyl-D-alanine cleaving peptidase (a highly penicillin-sensitive enzyme), the rate of enzyme acylation is not or only little affected when the oxyimino group is in the syn configuration, but is decreased when the oxyimino group is in the anti configuration; (iii) with the Actinomadura R39 serine D-alanyl-D-alanine-cleaving peptidase (an exceedingly highly penicillin-sensitive enzyme), the rate of enzyme acylation is unaffected whatever the configuration of the substituent. The oxidation of the sulphur atom of the dihydrothiazine ring on the beta-face of the molecule makes it both a poorer inactivator of the DD-peptidases and a poorer substrate of the beta-lactamase. The Streptomyces albus G Zn2+-containing D-alanyl-D-alanine-cleaving peptidase (a highly penicillin-resistant enzyme) remains highly resistant to all compounds tested

Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples

<p>Abstract</p> <p>Background</p> <p>Toxocarosis is a zoonotic disease caused by <it>Toxocara canis</it> (<it>T. canis</it>) and/or <it>Toxocara cati</it> (<it>T. cati</it>)<it>,</it> two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of <it>T. canis</it> or <it>T. cati</it>, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount.</p> <p>Methods</p> <p>A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of <it>T. canis</it> and <it>T. cati</it> eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including <it>T. canis</it>, <it>T. cati</it>, <it>T. leonina</it>, <it>Ascaris suum</it> (<it>A. suum</it>) and <it>Parascaris equorum</it> (<it>P. equorum</it>). The assay was used to assess the presence of <it>T. cati</it> eggs in several samples, including 12 clean soil samples spiked with eggs of either <it>T. cati</it> or <it>A. suum</it>, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination.</p> <p>Results</p> <p>2qPCR assay allowed specific detection of <it>T. canis</it> and <it>T. cati</it>, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces.</p> <p>Conclusion</p> <p>The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of <it>T.canis</it> and/or <it>T. cati</it> eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.</p