Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury. Conclusion In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

Color variation in museum specimens of birds: effects of stress, pigmentation, and duration of storage

Animal coloration often serves as a signal to others that may communicate traits about the individual such as toxicity, status, or quality. Colorful ornaments in many animals are often honest signals of quality assessed by mates, and different colors may beproduced by different biochemical pigments. Investigations of the mechanisms responsible for variation in color expression among birds are best when including a geographically and temporally broad sample. In order to obtain such a sample, studies such as this often use museum specimens; however, in order for museum specimens toserve as an accurate replacement, they must accurately represent living birds, or we must understand the ways in which they differ. In this thesis, I investigated the link between feather corticosterone, a hormone secreted in response to stress, and carotenoid-basedcoloration in the Red-winged Blackbird (Agelaius phoeniceus) in order to explore a mechanistic link between physiological state and color expression. Male Red-winged Blackbirds with lower feather corticosterone had significantly brighter red epaulets than birds with higher feather corticosterone, while I found no significant changes in red chroma. I also performed a methodological comparison of color change in museum specimens among different pigment types (carotenoid and psittacofulvin) and pigments in different locations in the body (feather and bill carotenoids) in order to quantify colorchange over time. Carotenoids and psittacofulvins showed significant reductions in red brightness and chroma over time in the collection, and carotenoid color changed significantly faster than psittacofulvin color. Both bill and feather carotenoids showed significant reductions in red brightness and red chroma over time, but change of both red chroma and red brightness occurred at a similar rate in feathers and bills. In order to use museum specimens of ecological research on bird coloration specimen age must be accounted for before the data can be used; however, once this is accomplished, museum- based color data may be used to draw conclusions about wild populations.

Distribution of mRNA coding for 5-hydroxytryptamine receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 7 subtypes of 5-hydroxytryptamine receptors (5-HTRs) in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation (CDD). SAMPLE POPULATION: Full-thickness intestinal wall biopsy specimens were obtained from the ileum, cecum, proximal loop of the ascending colon, and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy dairy cows allocated to 2 control groups (specimens collected during routine laparotomy [group 2] or after cows were slaughtered [group 3]). PROCEDURE: Amounts of mRNA coding for 7 subtypes of 5-HTRs (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, and 5-HT4) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed as the percentage of mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA coding for 5-HTR1B, 5-HTR2B, and 5-HTR4 was significantly lower in cows with CDD than in healthy cows. For 5-HTR2B and 5-HTR4, significant differences between cows with CDD and control cows were most pronounced for the ELSC. Expression of mRNA for 5-HTR1D, 5-HTR1F, and 5-HTR2A was extremely low in all groups, and mRNA for 5-HTR1A was not detected. CONCLUSIONS AND CLINICAL RELEVANCE: Relative concentrations of mRNA coding for 5-HTR1B, 5-HT2B, and 5-HTR4 were significantly lower in the intestines of cows with CDD than in the intestines of healthy dairy cows, especially for 5-HT2B and 5-HTR4 in the ELSC. This supports the hypothesis that serotonergic mechanisms, primarily in the spiral colon, are implicated in the pathogenesis of CDD.

Extended CT scale overcomes restoration caused streak artifacts for dental identification in CT--3D color encoded automatic discrimination of dental restorations

OBJECTIVE: Besides DNA, dental radiographs play a major role in the identification of victims in mass casualties or in corpses with major postmortem alterations. Computed tomography (CT) is increasingly applied in forensic investigations and is used to scan the dentition of deceased persons within minutes. We investigated different restoration materials concerning their radiopacity in CT for dental identification purposes. METHODS: Extracted teeth with different filling materials (composite, amalgam, ceramic, temporary fillings) were CT scanned. Radiopacities of the filling materials were analyzed in extended CT scale images. RESULTS: Radiopacity values ranged from 6000-8500HU (temporary fillings), 4500-17000HU (composite fillings) and >30710HU (Amalgam and Gold). The values were used to define presets for a 3D colored volume rendering software. CONCLUSIONS: The effects of filling material caused streak artifacts could be distinctively reduced for the assessment of the dental status and a postprocessing algorithm was introduced that allows for 3D color encoded visualization and discrimination of different dental restorations based on postmortem CT data.

Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene

The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.

Comparison of musical activities of cochlear implant users with different speech-coding strategies

Speech coding might have an impact on music perception of cochlear implant users. This questionnaire study compares the musical activities and perception of postlingually deafened cochlear implant users with three different coding strategies (CIS, ACE, SPEAK) using the Munich Music Questionnaire. Overall, the self-reported perception of music of CIS, SPEAK, and ACE users did not differ by very much.

The end1 gene of Schizosaccharomyces pombe coding for a DNase is identical with the pnu1 gene coding for an RNase

The DNA nuclease activity encoded by the end1 gene, and its inactivation by mutation, was described in connection with the characterization of DNA topoisomerases in the fission yeast Schizosaccharomyces pombe (Uemura and Yanagida, 1984). Subsequently, end1 mutant strains were used for the preparation of cell extracts for the study of enzymes and intermediates involved in DNA metabolism. The molecular identification of the end1 gene and its identity with the pnu1 gene is presented. The end1-458 mutation alters glycine to glutamate in the conserved motif TGPYLP. The pnu1 gene codes for an RNase that is induced by nitrogen starvation (Nakashima et al., 2002b). Thus, the End1/Pnu1 protein, like related mitochondrial proteins in other organisms, is an example of a sugar-non-specific nuclease. The analysis of strains carrying a pnu1 deletion revealed no defects in meiotic recombination and spore viability.