Heterogeneous molecular behavior in liver tumors (HCC and CCA) of two patients with acute intermittent porphyria.

INTRODUCTION: Acute intermittent porphyria (AIP) is a very rare (orphan) metabolic disorder of porphyrin biosynthesis which is characterized by elevated plasma and urine levels of 5-aminolevulinic acid (5-ALA) and porphobilinogen (PBG). Patients with this disorder which is caused by a germline mutation of the hydroxymethylbilan-synthase (HMBS)-gene have a high risk of primary liver cancer which may be determined by disease activity. The exact mechanism of carcinogenesis of this rare tumor is unknown, however. MATERIALS AND METHODS: We analyzed paraffin-embedded formalin-fixed liver tumor and normal liver specimens of two female AIP patients treated at the Munich EPNET center. One patient had developed hepatocellular carcinoma (HCC), the other intrahepatic cholangiocarcinoma (CCA). Since biallelic inactivation of HMBS had been observed in one study, we used Sanger and next-generation sequencing with a 8 gene porphyria panel plus 6 potential modifier loci to search for mutations in DNA extractions. RESULTS: In the patient with the HCC, we found a second inactivating mutation in the HMBS gene in the tumor but not in the adjacent normal liver tissue. No mutation could be found in the liver tissues of the patient with CCA, however. CONCLUSIONS: Biallelic inactivation of HMBS or protoporphyrinogen-oxidase (PPOX), another enzyme of porphyrin biosynthesis, has been observed in patients with acute porphyrias and liver tumors. We could confirm this in our patient with HCC with a mutation in HMBS but not in the one with CCA. Since 5-ALA can be converted into carcinogenic substances such as 4,5-dioxovaleric acid (DOVA) or 3,6-dihydropyrazine-2,5-dipropanoic acid (= cyclic dimerization product of 5-ALA), local production of these metabolites in hepatic areas with complete loss of HMBS activity may contribute to liver carcinogenesis.

Real-time quality control of optical backscattering data from Biogeochemical-Argo floats.

BACKGROUND: Biogeochemical-Argo floats are collecting an unprecedented number of profiles of optical backscattering measurements in the global ocean. Backscattering (BBP) data are crucial to understanding ocean particle dynamics and the biological carbon pump. Yet, so far, no procedures have been agreed upon to quality control BBP data in real time. METHODS: Here, we present a new suite of real-time quality-control tests and apply them to the current global BBP Argo dataset. The tests were developed by expert BBP users and Argo data managers and have been implemented on a snapshot of the entire Argo dataset. RESULTS: The new tests are able to automatically flag most of the "bad" BBP profiles from the raw dataset. CONCLUSIONS: The proposed tests have been approved by the Biogeochemical-Argo Data Management Team and will be implemented by the Argo Data Assembly Centres to deliver real-time quality-controlled profiles of optical backscattering. Provided they reach a pressure of about 1000 dbar, these tests could also be applied to BBP profiles collected by other platforms.

Further development of the beta-lactamase MutaGen assay and evaluation by comparison with Ames fluctuation tests and the umu test.

A rapid, high-throughput bacterial mutagenicity test system has been developed (MutaGen test) that detects reversions of inactivating base-pair substitutions and frameshifts in a TEM-1 class A beta-lactamase (ampicillinase) gene. To quickly and sensitively detect mutagens, the system utilises a series of plasmids that contain the mutated ampicillinase gene and the mucAB operon. Inactivating mutations in the ampicillinase gene include frameshifts integrated into repetitive GC-sequences and G-runs known to be mutagenic hot-spots, and base-pair substitutions inserted in or around the beta-lactamase active site. Frameshift mutations completely inactivated the enzyme only when located downstream of the active-site serine (Ser68). Previous (reporter gene based) assays with this system have detected reversion to ampicillin resistance by luminescence driven by induction of the tet-promotor controlled lacZ gene. In the present study, we describe the construction and evaluation of 19 additional potential tester strains. We also developed conditions for detecting reversions by pH shift using bromocresol purple and by directly detecting the enzymatic activity of beta-lactamase using nitrocefin. A 384-well format version of the pH shift MutaGen test was used to assay more than 20 chemicals. The responses in the assay were compared with responses for the same chemicals in the umu test and Ames fluctuation assays. The results indicate that the MutaGen test has high specificity for detecting specific mutations and, in some instances, better sensitivity than the other tests. Since the test is easy to conduct, sterile working conditions are not necessary, and the mutagenicity results are available either within one working day or overnight, the assay shows promise for the rapid screening of potentially genotoxic substances.

Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay).

The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity.

Effect of the silicone disclosing procedure on the shear bond strength of composite cements to ceramic restorations.

STATEMENT OF PROBLEM: There is no evidence-based information on how ceramic restorations with an adhesive bond between restoration material and composite cement may be influenced by a silicone disclosing agent. PURPOSE: The aim of this study was to determine the effects of the silicone disclosing procedure on the shear bond strength of composite cements in the luting of industrial sintered and laboratory sintered ceramic restorations. MATERIAL AND METHODS: Thirty standardized (15 x 10 x 9 mm) prefabricated ceramic specimens (Groups 1, 3, 5) and 30 standardized (15 x10 x 9 mm) conventionally sintered ceramic specimens (Groups 2, 4, 6) were roughened with sandpaper (800-grit). Each group contained 10 specimens. Groups 3 and 4 were conditioned with hydrofluoric acid and primed with silane solution after the use of a silicone disclosing procedure. Groups 1 and 2 served as the control groups, where no silicone disclosing procedure was performed. Groups 5 and 6 were insulated with glycerine before the silicone disclosing procedure. A glass tube (4.5 mm in diameter) was used to apply a cylinder of dual-polymerized composite cement to the conditioned surfaces. All specimens were submitted to 5000 thermocycles (5 degrees to 55 degrees C) to simulate the in vivo situation. The specimens were subjected to a shear-pull test at a constant crosshead speed of 5 mm/min with a universal testing machine. The comparative shear bond strengths were analyzed by use of Duncan's test (alpha=0.05). RESULTS: Shear bond strength values for Groups 1 (9.86 +/- 4.97 MPa) and 2 (9.56 +/- 4.47 Mpa) were obtained with no significant differences. Lower but significantly undifferent values were obtained for Groups 3 (7.49 +/- 4.67 MPa) and 4 (7.62 +/- 3.49 MPa) after the use of a silicone disclosing procedure. In Groups 5 (8.21 +/- 4.75 MPa) and 6 (8.22 +/- 3.59 MPa), including insulation with glycerine before the silicone disclosing procedure, no significant differences were obtained. CONCLUSION: Within the limitations of this study, the use of silicone disclosing procedures before conditioning the ceramic surface did not lead to a significant reduction of the shear bond strength between ceramic and composite cement. The ceramic materials used (industrial-sintered versus laboratory-sintered ceramic) had no significant influence on adhesion.