Highly Efficient Non-palladium Catalyzed Controlled Synthesis and X-ray Analysis of Functionalized 1,2-Diaryl-, 1,2,3-Triaryl- and 1,2,3,4-Tetraarylbenzene

A general, two-step highly efficient synthesis of 1,2-diaryl-, 1,2,3-triaryl- and 1,2,3,4-tetraarylbenzenes from simple stitching of alpha-oxo-ketene-S,S-acetals and active methylene compounds via a ‘lactone intermediate’ is described. This procedure offers easy access to highly functionalized arylated-benzenes containing sterically demanding groups in good to excellent yields. The novelty of the procedure lies in the fabrication of aromatic compounds with desired conformational flexibility along the molecular axis in a transition metal-free environment through easily accessible precursors. The crystal analysis of these arylated-benzene scaffolds showed that the peripheral aryl rings are arranged in propeller-like fashion with respect to the central benzene rings. Examination of the crystal packing in the structure of a 1,2,3,4-tetraarylbenzene 12c revealed a “N…pi interaction” between molecules related by a two-fold screw axis running in a direction. It is interesting that the repeat of the array of N…pi interaction around the axis of the 1,2,3,4-tetraarylbenzene 12c enforces the molecules in a helical pattern.

Effect of Feeding Procedure and Intake Level on Steer Feedlot Performance and Carcass Composition: A Progress Report

Two feedlot trials were conducted to evaluate the effects of feeding frequency (once daily in the morning, once daily in the afternoon or twice daily) and feeding level (ad libitum, 95% of ad libitum, or 90% of ad libitum) on the feedlot performance and carcass composition of beef steers. Data were collected using 196 yearling beef steers. In both trials cattle fed once daily in the morning tended to have higher gains and better feed efficiencies than cattle fed once daily in the afternoon or cattle fed twice daily. Overall, cattle restricted to 95% and 90% of ad libitum intake levels had better feed efficiencies than cattle with ad libitum access to feed. Cattle fed once daily in the morning tended to have less backfat than cattle fed once daily in the afternoon or cattle fed twice daily.

Repeat chlamydia screening among adolescents: cohort study in a school-based programme in New Orleans

OBJECTIVES To describe uptake of chlamydia screening, determine rates of repeated yearly screening and investigate determinants of repeated participation in an organised school-based screening programme. METHODS The authors analysed data from 1995 to 2005 from female and male students in up to 13 schools in New Orleans, Louisiana, USA. The authors calculated proportions of students tested among all enrolled students and among those with parental consent and the percentage of positive chlamydia tests in each school year. The authors used random effects logistic regression to examine the effect of past screening history on subsequent participation. RESULTS 35 041 students were registered for at least one school year. Overall coverage was >30% in all school years. Among all students registered for 4 years, 10.6% (95% CI 9.3% to 12.0%) of women and 12.7% (95% CI 11.2% to 14.2%) of men had a test every year. Among students with parental consent for 4 years, 49.3% (95% CI 44.6% to 54.1%) of women and 59.3% (95% CI 54.5% to 64.0%) of men had a test every year. Among students registered for 2 or more years, those with a previous positive chlamydia test were less likely to have a subsequent test (female adjusted OR 0.77, 95% CI 0.67 to 0.88 and male adjusted OR 0.84, 95% CI 0.69 to 1.02). Chlamydia positivity increased over time. CONCLUSIONS High levels of uptake can be achieved in school-based chlamydia screening programmes, but repeated yearly screening is difficult to sustain over time.

Catheter ablation of persistent and long-standing persistent atrial fibrillation. Strategies and results

Catheter ablation for paroxysmal atrial fibrillation is a meanwhile established therapy option, which is most frequently performed using radiofrequency ablation. Mid-term success rate of 70 % are achievable with a single ablation procedure. However, the mechanistics of persistent atrial fibrillation are less well understood and catheter ablation is a far more challenging procedure. Different ablation approaches are being performed to treat persistent atrial fibrillation ranging from sole pulmonary vein isolation to additional ablation of fractionated electrograms aiming for termination of atrial fibrillation. Thus far, it has not been investigated which strategy is most successful in treating persistent atrial fibrillation. After extended ablation of atrial fibrillation, occurrence of organized atrial arrhythmias is not uncommon and can be successfully ablated. These consecutive arrhythmias can be considered as a next step towards stable sinus rhythm after repeat ablation. Improvement of mapping methods as well as a better understanding of mechanisms of atrial fibrillation may increase success rate of catheter ablation of persistent atrial fibrillation and may also help to improve success rate of these complex procedures.

Fluid intake and all-cause mortality, cardiovascular mortality, and kidney function: a population-based longitudinal cohort study

BACKGROUND Drinking eight glasses of fluid or water each day is widely believed to improve health, but evidence is sparse and conflicting. We aimed to investigate the association between fluid consumption and long-term mortality and kidney function. METHODS We conducted a longitudinal analysis within a prospective, population-based cohort study of 3858 men and women aged 49 years or older residing in Australia. Daily fluid intake from food and beverages not including water was measured using a food frequency questionnaire. We did multivariable adjusted Cox proportional hazard models for all-cause and cardiovascular mortality and a boot-strapping procedure for estimated glomerular filtration rate (eGFR). RESULTS Upper and lower quartiles of daily fluid intake corresponded to >3 L and <2 L, respectively. During a median follow-up of 13.1 years (total 43 093 years at risk), 1127 deaths (26.1 per 1000 years at risk) including 580 cardiovascular deaths (13.5 per 1000 years at risk) occurred. Daily fluid intake (per 250 mL increase) was not associated with all-cause [adjusted hazard ratio (HR) 0.99 (95% CI 0.98-1.01)] or cardiovascular mortality [HR 0.98 (95% CI 0.95-1.01)]. Overall, eGFR reduced by 2.2 mL/min per 1.73 m(2) (SD 10.9) in the 1207 (31%) participants who had repeat creatinine measurements and this was not associated with fluid intake [adjusted regression coefficient 0.06 mL/min/1.73 m(2) per 250 mL increase (95% CI -0.03 to 0.14)]. CONCLUSIONS Fluid intake from food and beverages excluding water is not associated with improved kidney function or reduced mortality.

Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Penetrating or stricturing diseases are the major determinants of time to first and repeat resection surgery in Crohn's disease

BACKGROUND About 80% of patients with Crohn's disease (CD) require bowel resection and up to 65% will undergo a second resection within 10 years. This study reports clinical risk factors for resection surgery (RS) and repeat RS. METHODS Retrospective cohort study, using data from patients included in the Swiss Inflammatory Bowel Disease Cohort. Cox regression analyses were performed to estimate rates of initial and repeated RS. RESULTS Out of 1,138 CD cohort patients, 417 (36.6%) had already undergone RS at the time of inclusion. Kaplan-Meier curves showed that the probability of being free of RS was 65% after 10 years, 42% after 20 years, and 23% after 40 years. Perianal involvement (PA) did not modify this probability to a significant extent. The main adjusted risk factors for RS were smoking at diagnosis (hazard ratio (HR) = 1.33; p = 0.006), stricturing with vs. without PA (HR = 4.91 vs. 4.11; p < 0.001) or penetrating disease with vs. without PA (HR = 3.53 vs. 4.58; p < 0.001). The risk factor for repeat RS was penetrating disease with vs. without PA (HR = 3.17 vs. 2.24; p < 0.05). CONCLUSION The risk of RS was confirmed to be very high for CD in our cohort. Smoking status at diagnosis, but mostly penetrating and stricturing diseases increase the risk of RS.

Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)(240) insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

14-3-3 proteins interact with the beta-thymosin repeat protein Csp24.

Conditioned stimulus pathway protein 24 (Csp24) is a beta-thymosin-like protein that is homologous to other members of the family of beta-thymosin repeat proteins that contain multiple actin binding domains. Actin co-precipitates with Csp24 and co-localizes with it in the cytosol of type-B photoreceptor cell bodies. Several signal transduction pathways have been shown to regulate the phosphorylation of Csp24 and contribute to cellular plasticity. Here, we report the identification of the adapter protein 14-3-3 in lysates of the Hermissenda circumesophageal nervous system and its interaction with Csp24. Immunoprecipitation experiments using an antibody that is broadly reactive with several isoforms of the 14-3-3 family of proteins showed that Csp24 co-precipitates with 14-3-3 protein, and nervous systems stimulated with 5-HT exhibited a significant increase in co-precipitated Csp24 probed with a phosphospecific antibody as compared with controls. These results indicate that post-translational modifications of Csp24 regulate its interaction with 14-3-3 protein, and suggest that this mechanism may contribute to the control of intrinsic enhanced excitability.

ANKRD1, the gene encoding cardiac ankyrin repeat protein, is a novel dilated cardiomyopathy gene.

OBJECTIVES: We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. BACKGROUND: CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. METHODS: In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. CONCLUSIONS: ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

Mapping and cloning of genes from portions of the human genome using somatic cell hybrids

Human x rodent somatic cell hybrids have played an important role in human genetics research. They have been especially useful for assigning genes to chromosomes and isolating DNA markers from specific regions of the human genome.^ By employing a combination of somatic cell genetic, recombinant DNA, and cytogenetic techniques, human DNA excision repair gene ERCC4 was mapped regionally to human 16p13.13-13.2, even though the gene has not been cloned. Human x Chinese hamster ovary (CHO) cell hybrids selected for human ERCC4 activity and containing 16p13.1-p13.3 as the only human genetic material were identified. These hybrids were used to order DNA markers located in 16p13.1-p13.3. New DNA markers physically close to ERCC4 were isolated from such hybrids. Using amplified human DNA from the hybrids as probe in fluorescent in situ hybridization, the short arm breakpoint in the chromosome 16 inversion associated with acute myelomonocytic leukemia (AMML) was found to be physically close to the ERCC4 gene. The physical mapping and eventually, the cloning of the ERCC4 gene, will benefit the understanding of the DNA repair system and the study of other important biomedical problems such as tumorigenesis.^ To facilitate the cloning of ERCC4 gene and, in general, the cloning of genes from any defined regions of the human genome, a method was developed for the direct isolation of human transcribed genes ffom somatic cell hybrids. cDNA was prepared from human x rodent hybrid by using consensus 5$\sp\prime$ splice site sequences as primers. These primers were designed to select immature, unspliced messenger RNA (still retaining species specific repeat sequences) as templates. Screening of a derived cDNA library for human repeat sequences resulted in the isolation of human clones at the anticipated frequency with characteristics expected of exons of transcribed human genes. The usefulness of the splice site specific primers was analyzed and the cDNA synthesis conditions with these primers were optimized. The procedure was shown to be sensitive enough to clone weakly expressed genes. Studying the expression of the represented genes with the isolated clones was shown to be feasible. Such regional specific human gene fragments will be very valuable for many human genetic studies such as the search of inherited disease genes and the construction of a cDNA map of the human genome. ^