Spin-crossover in metallomesogens of iron(II)

Covalent grafting mesogenic groups to the coordination cores of the parent mononuclear low-spin and spin-crossover compounds afforded metallomesogenic complexes of iron(II). In comparison with the parent complexes the spin-crossover properties of the alkylated derivatives are substantially modified. The type of the modification was found to be dependent on the properties of the parent system and the nature of the used anion, however, the general tendency is the destabilization of the low-spin state at the favor of spin-crossover or high-spin behavior below 400 K. The structural insight revealed the micro-segregated layered organization. The effect of the alkylation of the parent compounds consists first of all in the change of the lattice to a two-dimensional lamellar one retaining significant intermolecular contacts only within the ionic bilayers. The comprehensive analysis of the structural and thermodynamic data in the homologous series pointed at the mechanism of the interplay between the structural modification on melting and the induced anomalous change of the magnetic properties. A family of one-dimensional spin-crossover polymers was synthesized and characterized using a series of spectroscopic methods, X-ray powder diffraction, magnetic susceptibility measurements and differential scanning calorimetry. The copper analogue of was also synthesized and its crystal structure solved. In comparison with the mononuclear systems, the polymeric mesogens of iron(II) are less sensitive to the glass transition, which was attributed to the moderate concomitant variation of the structure. Nevertheless, the observed increase of the magnetic hysteresis with lengthening of the alkyl substituents was ascribed to the interplay of the structural reorganization of the coordination core due to spin-crossover with the structural delay in the spatial reorganization of the mesogenic substituents. The classification of mononuclear and polymeric metallomesogens according to the interactions between the structural- and the spin-transition and analysis of the data on the reported spin-crossover metallomesogens led to the separation of three types, namely: Type i: systems with coupling between the electronic structure of the iron(II) ions and the mesomorphic behavior of the substance; Type ii: systems where both transitions coexist in the same temperature region but are not coupled due to competition with the dehydration or due to negligible structural transformation; Type iii: systems where both transitions occur in different temperature regions and therefore are uncoupled. Fine-tuning, in particular regarding the temperature at which the spin-transition occurs with hysteresis properties responsible for the memory effect, are still a major challenge towards practical implementation of spin-crossover materials. A possible answer to the problem could be materials in which the spin-crossover transition is coupled with another transition easily controllable by external stimuli. In the present thesis we have shown the viability of the approach realized in the mesogenic systems with coupled phase- and spin-transitions.

Exploring the biofilm of Streptococcus agalactiae to identify virulence factors

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is the primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30-76% of the cases of neonatal meningitis. Biofilms are dense aggregates of surface-adherent microorganisms embedded in an exopolysaccharide matrix. Centers for Disease Control and Prevention estimate that 65% of human bacterial infections involve biofilms (Post et al., 2004). In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and/or virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low- and non- biofilm forming strains and reduce ambiguous data interpretation. This protocol was applied to screen the in vitro biofilm formation ability of more than 350 GBS clinical isolates from pregnant women and neonatal infections belonging to different serotype, in relation to media composition and pH. The results showed the enhancement of GBS biofilm formation in acidic condition and identified a subset of isolates belonging to serotypes III and V that forms strong biofilms in these conditions. Interestingly, the best biofilm formers belonged to the serotype III hypervirulent clone ST-17.It was also found that pH 5.0 induces down-regulation of the capsule but that this reduction is not enough by itself to ensure biofilm formation. Moreover, the ability of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm formation and contribute to the biofilm structural stability. Finally, a set of proteins potentially expressed during the GBS in vitro biofilm formation were identified by mass spectrometry.

Untersuchungen zur Funktion multipler DnaJ-Proteine in dem Cyanobakterium Synechocystis sp. PCC 6803

Sowohl in Synechocystis sp. PCC 6803 als auch in anderen Cyanobakterien konnten multiple DnaJ-Proteine nachgewiesen werden, deren Funktion jedoch noch weitestgehend unverstanden ist. Im Rahmen dieser Arbeit wurden die Funktionen der multiplen DnaJ-Proteine von Synechocystis sp. charakterisiert. Das DnaJ-Protein, Sll0897 gehört aufgrund seiner Domänenstruktur zu den Typ I-Proteinen, Slr0093 und Sll1933 zu den Typ II-Proteinen und Sll0909, Sll1011, Sll1384 und Sll1666 zu den Typ III DnaJ-Proteinen. Durch Komplementationsstudien des E. coli ΔdnaJ-Stammes OD259 konnte eine Komplementation des Wachstumsdefekts bei höheren Temperaturen durch die Proteine Slr0093 und Sll0897 gezeigt werden. In Synechocystis war eine komplette Disruption von sll1933 nicht möglich, weshalb das Protein Sll1933 unter normalen Wachstumsbedingungen essentiell ist. Doppelte Insertionmutationen waren lediglich bei der Kombination der Gene sll0909 und sll1384 möglich. Untersuchungen des Wachstumsverhaltens der dnaJ-Disruptions-stämme unter Hitze- und Kältestressbedingungen zeigten, dass das Protein Sll0897 eine wichtige Funktion bei der Stressantwort in Synechocystis besitzt und unter Hitzestressbedingungen essentiell ist. Eine vollständige Deletion des Gens sll0897 war Synechocystis sp. bereits unter normalen Wachstumsbedingungen nicht möglich. Bei den für ein Wachstum mindestens notwendigen Domänen des Sll0897 handelt es sich um die charakteristische J-Domäne und die Glycin-Phenylalanin-reiche Domäne. Unter Hitzestressbedingungen ist das Volllängen-Protein Sll0897 für ein Wachstum essentiell. rnNeben den in vivo Wachstumsexperimenten wurde eine Methode zur heterologen Expression der sieben DnaJ-Proteine in E. coli und einer nativen Reinigung von Slr0093, Sll0897, Sll0909 und Sll1666 etabliert. Untersuchungen zur Thermostabilität der gereinigten Proteine zeigten für das Slr0093 und Sll1666 einen reversiblen Prozess, wodurch sie auch nach dem Hitzestress noch als Faltungshelfer fungieren können. Bei den Proteinen Sll0897 und Sll0909 ist der Prozess jedoch nicht reversibel, so dass sie nach Hitzestresseinwirkung neu synthetisiert oder durch Chaperoneinwirkung korrekt gefaltet werden müssen. Die Affinitäts-„Pull-Down“ Analysen lieferten keine klaren Hinweise auf die DnaK-Interaktionspartner der Proteine Slr0093, Sll0897, Sll0909 und Sll1666, weshalb weitere Untersuchungen notwendig sind. Mit Hilfe der Gelfiltrationsanalysen konnten die errechneten molaren Massen der Proteine Slr0093 und Sll1666 bestätigt und beide Proteine in einer monomeren Form nachgewiesen werden. Die DnaJ-Proteine Sll0897 und Sll0909 konnten in zwei oligomeren Zuständen detektiert werden. Analysen der ATPase-Aktivität des DnaK2-Proteins alleine und des DnaK2-Proteins zusammen mit den DnaJ-Proteinen Slr0093, Sll0897, Sll0909 und Sll1666 zeigten eine Steigerung der ATP-Hydrolyserate bei der Interaktion von DnaK und DnaJ, wobei Sll0897 die größte Steigerung der ATPase-Aktivität des DnaK2 induzierte.

Specific processing of tenascin-C by the metalloprotease meprinbeta neutralizes its inhibition of cell spreading

The metalloprotease meprin has been implicated in tissue remodelling due to its capability to degrade extracellular matrix components. Here, we investigated the susceptibility of tenascin-C to cleavage by meprinbeta and the functional properties of its proteolytic fragments. A set of monoclonal antibodies against chicken and human tenascin-C allowed the mapping of proteolytic fragments generated by meprinbeta. In chicken tenascin-C, meprinbeta processed all three major splicing variants by removal of 10kDa N-terminal and 38kDa C-terminal peptides, leaving a large central part of subunits intact. A similar cleavage pattern was found for large human tenascin-C variant where two N-terminal peptides (10 or 15kDa) and two C-terminal fragments (40 and 55kDa) were removed from the intact subunit. N-terminal sequencing revealed the exact amino acid positions of cleavage sites. In both chicken and human tenascin-C N-terminal cleavages occurred just before and/or after the heptad repeats involved in subunit oligomerization. In the human protein, an additional cleavage site was identified in the alternative fibronectin type III repeat D. Whereas all these sites are known to be attacked by several other proteases, a unique cleavage by meprinbeta was located to the 7th constant fibronectin type III repeat in both chicken and human tenascin-C, thereby removing the C-terminal domain involved in its anti-adhesive activity. In cell adhesion assays meprinbeta-digested human tenascin-C was not able to interfere with fibronectin-mediated cell spreading, confirming cleavage in the anti-adhesive domain. Whereas the expression of meprinbeta and tenascin-C does not overlap in normal colon tissue, inflamed lesions of the mucosa from patients with Crohn's disease exhibited many meprinbeta-positive leukocytes in regions where tenascin-C was strongly induced. Our data indicate that, at least under pathological conditions, meprinbeta might attack specific functional sites in tenascin-C that are important for its oligomerization and anti-adhesive activity.

On-table reconstruction of comminuted fractures of the radial head

The most widely accepted treatment for comminuted fractures of the radial head is either the excision or open reduction and internal fixation. The purpose of the present study is to evaluate the value of an 'on-table' reconstruction technique in severely comminuted fractures of the radial head. In this study, two patients with a Mason type-III and four patients with a Mason type-IV radial-head fracture were treated with 'on-table' reconstruction and fixation using low-profile mini-plates. After a mean follow-up of 112 months (47-154 months), the mean elbow motion was 0-6-141 degrees extension flexion with 79 degrees of pronation and 70 degrees of supination. The mean Broberg and Morrey functional rating score was 97.0 points, the Mayo Elbow Performance Index was 99.2 points and the mean Disabilities of the Arm, Shoulder, and Hand (DASH) Outcome Measure score was 1.94 points. One patient had symptoms of degenerative changes, with a slight joint-space narrowing. There were no radiographic signs of devitalisation at final examination. Comminuted fractures of the radial head, which would otherwise require excision, can be successfully treated with an 'on-table' reconstruction technique.

Comminuted fractures of the radial head and neck: is fixation to the shaft necessary?

Mason type III fractures of the radial head are treated by open reduction and internal fixation, resection or prosthetic joint replacement. When internal fixation is performed, fixation of the radial head to the shaft is difficult and implant-related complications are common. Furthermore, problems of devascularisation of the radial head can result from fixation of the plate to the radial neck. In a small retrospective study, the treatment of Mason type III fractures with fixation of the radial neck in 13 cases (group 2) was compared with 12 cases where no fixation was performed (group 1). The mean clinical and radiological follow-up was four years (1 to 9). The Broberg-Morrey index showed excellent results in both groups. Degenerative radiological changes were seen more frequently in group 2, and removal of the implant was necessary in seven of 13 cases. Post-operative evaluation of these two different techniques revealed similar ranges of movement and functional scores. We propose that anatomical reconstruction of the radial head without metalwork fixation to the neck is preferable, and the outcome is the same as that achieved with the conventional technique. In addition degenerative changes of the elbow joint may develop less frequently, and implant removal is not necessary.

Classification of suppressor additives based on synergistic and antagonistic ensemble effects

Three fundamental types of suppressor additives for copper electroplating could be identified by means of potential Transient measurements. These suppressor additives differ in their synergistic and antagonistic interplay with anions that are chemisorbed on the metallic copper surface during electrodeposition. In addition these suppressor chemistries reveal different barrier properties with respect to cupric ions and plating additives (Cl, SPS). While the type-I suppressor selectively forms efficient barriers for copper inter-diffusion on chloride-terminated electrode surfaces we identified a type-II suppressor that interacts non-selectively with any kind of anions chemisorbed on copper (chloride, sulfate, sulfonate). Type-I suppressors are vital for the superconformal copper growth mode in Damascene processing and show an antagonistic interaction with SPS (Bis-Sodium-Sulfopropyl-Disulfide) which involves the deactivation of this suppressor chemistry. This suppressor deactivation is rationalized in terms of compositional changes in the layer of the chemisorbed anions due to the competition of chloride and MPS (Mercaptopropane Sulfonic Acid) for adsorption sites on the metallic copper surface. MPS is the product of the dissociative SPS adsorption within the preexisting chloride matrix on the copper surface. The non-selectivity in the adsorption behavior of the type-II suppressor is rationalized in terms of anion/cation pairing effects of the poly-cationic suppressor and the anion-modified copper substrate. Atomic-scale insights into the competitive Cl/MPS adsorption are gained from in situ STM (Scanning Tunneling Microscopy) using single crystalline copper surfaces as model substrates. Type-III suppressors are a third class of suppressors. In case of type-land type-II suppressor chemistries the resulting steady-state deposition conditions are completely independent on the particular succession of additive adsorption. In contrast to that a strong dependence of the suppressing capabilities on the sequence of additive adsorption ("first comes, first serves" principle) is observed for the type-IIIsuppressor. This behavior:is explained by a suppressor barrier that impedes not only the copper inter-diffusion but also the transport of other additives (e.g. SPS) to the copper surface. (C) 2011 Elsevier Ltd. All rights reserved.

A nonsense mutation in the optic atrophy 3 gene (OPA3) causes dilated cardiomyopathy in Red Holstein cattle

Cardiomyopathies are severe degenerative disorders of the myocardium that lead to heart failure. During the last three decades bovine dilated cardiomyopathy (BDCMP) was observed worldwide in cattle of Holstein-Friesian origin. In the Swiss cattle population BDCMP affects Fleckvieh and Red Holstein breeds. The heart of affected animals is enlarged due to dilation of both ventricles. Clinical signs are caused by systolic dysfunction and affected individuals die as a result of severe heart insufficiency. BDCMP follows an autosomal recessive pattern of inheritance and the disease-causing locus was mapped to bovine chromosome 18 (BTA18). In the present study we describe the successful identification of the causative mutation in the OPA3 gene located on BTA18 that was previously reported to cause 3-methylglutaconic aciduria type III in Iraqi-Jewish patients. We demonstrated conclusive genetic and functional evidence that the nonsense mutation c.343C>T in the bovine OPA3 gene causes the late-onset dilated cardiomyopathy in Red Holstein cattle.

Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study

OBJECTIVES: The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. METHODS: A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. RESULTS: Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n=2), IV (n=6), V (n=14) or VII-241 (n=4) or were non-typeable (n=2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated beta-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia] (88.3%), kanamycin [aph(3')-III] (90.3%), streptomycin [ant(6')-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (cat(pC221)) (57.3%) and rifampicin (1.9%). CONCLUSIONS: Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.

Genetic variations in IL28B and allergic disease in children

Environmental changes affecting the relationship between the developing immune system and microbial exposure have been implicated in the epidemic rise of allergic disease in developed countries. While early developmental differences in T cell function are well-recognised, there is now emerging evidence that this is related to developmental differences in innate immune function. In this study we sought to examine if differences associated with innate immunity contribute to the altered immune programming recognised in allergic children. Here, we describe for the first time, the association of carriage of the T allele of the tagging single nucleotide polymorphism rs12979860 3 kb upstream of IL28B, encoding the potent innate immune modulator type III interferon lambda (IFN-λ3), and allergy in children (p = 0.004; OR 4.56). Strikingly, the association between rs12979860 genotype and allergic disease is enhanced in girls. Furthermore, carriage of the T allele at rs12979860 correlates with differences in the pro-inflammatory profile during the first five years of life suggesting this contributes to the key differences in subsequent innate immune development in children who develop allergic disease. In the context of rising rates of disease, these immunologic differences already present at birth imply very early interaction between genetic predisposition and prenatal environmental influences.

Occurrence of gustducin-immunoreactive cells in von Ebner's glands of guinea pigs

An immunohistochemical examination of guinea-pig taste buds in vallate papillae revealed gustducin-immunoreactive cells in the area of von Ebner’s glands, minor salivary glands. Since there have been no reports describing those cells in these locations for other species, we investigated these glands in order both to localize the cells and compare their immunoreactive characteristics with corresponding cells in the vallate taste buds. The gustducin-immunoreactive cells coincided with cells containing no secretory granules in the end portion of the glands, which was supported by the electron-microscopic immunocytochemistry. Double immunofluorescence microscopy confirmed these cells to be entirely immunopositive to type III inositol 1,4,5-triphosphate receptor (IP3R-3), phospholipase Cβ2 (PLCβ2), and villin and also partly immunopositive to neuron-specific enolase (NSE) and calbindin D-28K. The gustducin-immunoreactive cells in the vallate taste buds exhibited completely the same immunoreactivities for these five molecules. Accordingly, the present results give credence to a consideration that the gustducin-immunnoreactive cells in both locations are identical in function(s) e.g., chemo-reception.

Impaired innate interferon induction in severe therapy resistant atopic asthmatic children

Deficient type I interferon-β and type III interferon-λ induction by rhinoviruses has previously been reported in mild/moderate atopic asthmatic adults. No studies have yet investigated if this occurs in severe therapy resistant asthma (STRA). Here, we show that compared with non-allergic healthy control children, bronchial epithelial cells cultured ex vivo from severe therapy resistant atopic asthmatic children have profoundly impaired interferon-β and interferon-λ mRNA and protein in response to rhinovirus (RV) and polyIC stimulation. Severe treatment resistant asthmatics also exhibited increased virus load, which negatively correlated with interferon mRNA levels. Furthermore, uninfected cells from severe therapy resistant asthmatic children showed lower levels of Toll-like receptor-3 mRNA and reduced retinoic acid inducible gene and melanoma differentiation-associated gene 5 mRNA after RV stimulation. These data expand on the original work, suggesting that the innate anti-viral response to RVs is impaired in asthmatic tissues and demonstrate that this is a feature of STRA.

Vascular endothelial cell-specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development

VE-PTP, a receptor-type phosphotyrosine phosphatase, associates with the tyrosine kinase receptor Tie-2 and VE-cadherin and enhances the adhesive function of the latter. Here, VE-PTP was found to be restricted to endothelial cells, with a preference for arterial endothelium. Mutant mice expressing a truncated, secreted form of VE-PTP lacking the cytoplasmic and transmembrane domains and the most membrane-proximal extracellular fibronectin type III repeat, showed severe vascular malformations causing lethality at 10 days of gestation. Although blood vessels were initially formed, the intraembryonic vascular system soon deteriorated. Blood vessels in the yolk sac developed into dramatically enlarged cavities. In explant cultures of mutant allantoides, endothelial cells were found next to vessel structures growing as cell layers. No signs for enhanced endothelial apoptosis or proliferation were observed. Thus, the activity of VE-PTP is not required for the initial formation of blood vessels, yet it is essential for their maintenance and remodeling.

Unveiling electrotransformation of Moraxella catarrhalis as a process of natural transformation

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhalis.

Development of a genotyping microarray for Usher syndrome

BACKGROUND: Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. METHODS: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. RESULTS: Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. CONCLUSION: The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.