Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase

Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase [4]. During DNA replication, cohesin establishes cohesion [5], and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin [4]. In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation [6]. How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion [7]. To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.     

Eya1 is required for the morphogenesis of mammalian thymus,parathyroid and thyroid

Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system. We have now examined the role of Eya1 during the morphogenesis of organs derived from the pharyngeal region, including thymus, parathyroid and thyroid. The thymus and parathyroid are derived from 3rd pharyngeal pouches and their development is initiated via inductive interactions between neural crest-derived arch mesenchyme, pouch endoderm, and possibly the surface ectoderm of 3rd pharyngeal clefts. Eya1 is expressed in all three cell types during thymus and parathyroid development from E9.5 and the organ primordia for both of these structures failed to form in Eya1(-/-) embryos. These results indicate that Eya1 is required for the initiation of thymus and parathyroid gland formation. Eya1 is also expressed in the 4th pharyngeal region and ultimobranchial bodies. Eya1(-/-) mice show thyroid hypoplasia, with severe reduction in the number of parafollicular cells and the size of the thyroid lobes and lack of fusion between the ultimobranchial bodies and the thyroid lobe. These data indicate that Eya1 also regulates mature thyroid gland formation. Furthermore, we show that Six1 expression is markedly reduced in the arch mesenchyme, pouch endoderm and surface ectoderm in the pharyngeal region of Eya1(-/-) embryos, indicating that Six1 expression in those structures is Eya1 dependent. In addition, we show that in Eya1(-/-) embryos, the expression of Gcm2 in the 3rd pouch endoderm is undetectable at E10.5, however, the expression of Hox and Pax genes in the pouch endoderm is preserved at E9.5-10.5. Finally, we found that the surface ectoderm of the 3rd and 4th pharyngeal region show increased cell death at E10.5 in Eya1(-/-) embryos. Our results indicate that Eya1 controls critical early inductive events involved in the morphogenesis of thymus, parathyroid and thyroid.     

Post-pandemic seroprevalence of pandemic influenza A (H1N1) 2009 infection (swine flu) among children <18 years in Germany

Background

We determined antibodies to the pandemic influenza A (H1N1) 2009 virus in children to assess: the incidence of (H1N1) 2009 infections in the 2009/2010 season in Germany, the proportion of subclinical infections and to compare titers in vaccinated and infected children.

Methodology/Principal Findings

Eight pediatric hospitals distributed over Germany prospectively provided sera from in- or outpatients aged 1 to 17 years from April 1^st to July 31^st 2010. Vaccination history, recall of infections and sociodemographic factors were ascertained. Antibody titers were measured with a sensitive and specific in-house hemagglutination inhibition test (HIT) and compared to age-matched sera collected during 6 months before the onset of the pandemic in Germany. We analyzed 1420 post-pandemic and 300 pre-pandemic sera. Among unvaccinated children aged 1–4 and 5–17 years the prevalence of HI titers (≥1∶10) was 27.1% (95% CI: 23.5–31.3) and 53.5% (95% CI: 50.9–56.2) compared to 1.7% and 5.5%, respectively, for pre-pandemic sera, accounting for a serologically determined incidence of influenza A (H1N1) 2009 during the season 2009/2010 of 25,4% (95% CI : 19.3–30.5) in children aged 1–4 years and 48.0% (95% CI: 42.6–52.0) in 5–17 year old children. Of children with HI titers ≥1∶10, 25.5% (95% CI: 22.5–28.8) reported no history of any infectious disease since June 2009. Among vaccinated children, 92% (95%-CI: 87.0–96.6) of the 5–17 year old but only 47.8% (95%-CI: 33.5–66.5) of the 1–4 year old children exhibited HI titers against influenza A virus (H1N1) 2009.

Conclusion

Serologically determined incidence of influenza A (H1N1) 2009 infections in children indicates high infection rates with older children (5–17 years) infected twice as often as younger children. In about a quarter of the children with HI titers after the season 2009/2010 subclinical infections must be assumed. Low HI titers in young children after vaccination with the AS03_B-adjuvanted split virion vaccine need further scrutiny.     

Pneumocystis carinii shows DNA homology with the ustomycetous red yeast fungi

Pneumocystis carinii causes life-threatening pneumonia in T-lymphocyte-immunodeficient subjects in transplant and oncology units or with acquired immune deficiency syndrome (AIDS). Recent DNA homology studies show P. carinii to be a fungus. To investigate the biology and epidemiology of this parasite further, we elected to determine for it a more precise taxonomic assignment within the fungal kingdom. We screened a wide range of organisms representing the major orders of fungi using DNA amplification and subsequently sequenced a portion of the mitochondrial gene encoding the large subunit ribosomal RNA. Our data show that the opportunistic pulmonary pathogen P. carinii is closely related to the ustomycetous red yeast fungi, a group which includes organisms that are extensively distributed throughout the environment and which release many widely dispersed airborne spores.     

Topology of the anion-selective porin Omp32 from Comamonas acidovorans.

Limited proteolysis experiments were performed with outer membranes from Comamonas acidovorans to probe the topology of its major protein component, the anion-selective porin Omp32. Proteinase K treatment above a critical temperature of 42 degrees C cleaved the surface-exposed regions of the porin, yielding membrane-embedded fragments which were separated by SDS polyacrylamide gel electrophoresis or reversed phase chromatography. The identification of the proteinase K-sensitive sites was performed by microsequencing. This allowed us to determine six surface-exposed sites of the porin, all located in nonconserved primary structure regions. These results along with the previously determined amino acid sequence and in conjunction with some structural constraints applicable to porins allowed us to propose a chain-folding model of the Omp32 porin. The features of our model are compared with the structure of the Rhodobacter capsulatus porin, recently established by X-ray crystallography (Weiss et al., 1991) and they are used to elucidate the structural basis of the anion selectivity.     

The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.

We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport.     

Rapid and sensitive assay for the phytotoxin rhizobitoxine.

Rhizobitoxine is a phytotoxin synthesized by some strains of the legume symbiont genus Bradyrhizobium and the plant pathogen Pseudomonas andropogonis. We demonstrate here a new enzymatic assay which is 100-fold more sensitive than previous assays and can detect as little as 1.0 pmol of rhizobitoxine. The assay is based on the inhibition of Salmonella typhimurium beta-cystathionase by rhizobitoxine. Interestingly, beta-cystathionase from Bradyrhizobium japonicum is insensitive to rhizobitoxine at concentrations lower than 75 microM.     

A synthetic fiber-reinforced stentless heart valve

There is strong evidence that failure of bioprosthetic and synthetic valves occurs as a consequence of high tensile and bending stresses, acting on the leaflets during opening and closing. In stented prostheses, whether synthetic or biological, the absence of contraction of the aortic base causes the leaflets to be subjected to an unphysiological degree of flexure, which is also related to calcification. However, a stentless synthetic valve, which has a flexible aorta base, can be a good alternative for stented synthetic valves. Moreover, fiber-reinforcement is assumed to lead to a decrease of tears and perforation as a result of reduced stresses in the weaker parts of the leaflets in their closed configuration. The manufacturing method for a stentless, fiber-reinforced, synthetic valve is presented. Prototypes are tested in a pulse duplicator system. The results show that the mean systolic pressure difference is very low, while the high regurgitation (up to 26%) is probably caused by a too small coaptation area of the leaflets.     

Improved production and function of llama heavy chain antibody fragments by molecular evolution

The aim of this study was to improve production level of llama heavy chain antibody fragments (V_HH) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V_HH fragments specific for the azo-dye reactive red-6. In the DNA shuffling process, three parental llama V_HH with high amino acid sequence identity with significant differences in production and functional characteristics were used. From these parental sequences, a S. cerevisiae library was created and 16 antigen specific shuffled V_HH fragments were selected. We found that these shuffled V_HH fragments were, (i) unique in sequence; (ii) composed of two or three parental sequences; (iii) in three V_HHs point mutations occurred; and (iv) antigen specificity was not changed. The four highest producers in the yeast S. cerevisiae were selected and production, affinity, and antigen binding at 90°C were compared with parental V_HHs. One shuffled V_HH was enhanced both in production (3.4-fold) and affinity (four-fold). A second shuffled V_HH displayed increased production (1.9-fold), and improved stability (2.4-fold) in antigen binding at 90°C. Structural analysis suggested that improved antigen binding is associated with the A24→V24 substitution, which reduces the size of the hydrophobic pit at the llama V_HH surface. We demonstrate that it is possible to improve desired characteristics of the same V_HH fragment simultaneously using DNA shuffling. Finally, this is one of the first examples of DNA shuffling improving temperature stability of an antibody fragment.