Physosmotic Induction of Chondrogenic Maturation Is TGF-β Dependent and Enhanced by Calcineurin Inhibitor FK506

Increasing extracellular osmolarity 100 mOsm/kg above plasma level to the physiological levels for cartilage induces chondrogenic marker expression and the differentiation of chondroprogenitor cells. The calcineurin inhibitor FK506 has been reported to modulate the hypertrophic differentiation of primary chondrocytes under such conditions, but the molecular mechanism has remained unclear. We aimed at clarifying its role. Chondrocyte cell lines and primary cells were cultured under plasma osmolarity and chondrocyte-specific in situ osmolarity (+100 mOsm, physosmolarity) was increased to compare the activation of nuclear factor of activated T-cells 5 (NFAT5). The effects of osmolarity and FK506 on calcineurin activity, cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling were analyzed using biochemical, gene, and protein expression, as well as reporter and bio-assays. NFAT5 translocation was similar in chondrocyte cell lines and primary cells. High supraphysiological osmolarity compromised cell proliferation, while physosmolarity or FK506 did not, but in combination increased proteoglycan and collagen expression in chondrocytes in vitro and in situ. The expression of the TGF-β-inducible protein TGFBI, as well as chondrogenic (SOX9, Col2) and terminal differentiation markers (e.g., Col10) were affected by osmolarity. Particularly, the expression of minor collagens (e.g., Col9, Col11) was affected. The inhibition of the FK506-binding protein suggests modulation at the TGF-β receptor level, rather than calcineurin-mediated signaling, as a cause. Physiological osmolarity promotes terminal chondrogenic differentiation of progenitor cells through the sensitization of the TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 further enhances signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our results help explain earlier findings and potentially benefit future cell-based cartilage repair strategies.     

Procedural influence on the properties of particleboards made from AKD modified chips

Wood chips were treated with alkyl ketene dimer (AKD) using three different processes to impart water resistance to particleboards. In the first process, AKD was blended with UF resin. Thickness swelling and water uptake after a 24 h immersion period (20 and 69%) were lower than in the control boards (28 and 81%) but were higher than in the paraffin references (10 and 22%). In process 2, AKD and UF resin were sprayed separately on the chips resulting in a greater reduction of thickness swelling (15%) and water uptake (49%) than in process 1. Paraffin references revealed a thickness swelling and water uptake of 7 and 25%, respectively. An extension of the pressing time in processes 1 and 2 did not increase water repellence. In process 3, particleboards were made from AKD-treated chips that were cured at 130 °C (24 h) prior to gluing. They showed a thickness swelling of 7% and a water uptake of 25%, whereas particleboards with paraffin exhibited levels of 8 and 29%. The thickness swelling and water uptake of boards with AKD increased when the curing time was reduced from 24 to 12 to 6 h (130 °C). Changing the curing temperature from 130 to 100 °C (12 h) had no effect on board properties. The IB of boards made from pre-cured chips with AKD (24 h/130 °C) was 44% lower than in controls and 35% lower than in paraffin references. This indicates that AKD impedes the adhesion.     

Probiotic lactobacilli and bifidobacteria in a fermented milk product with added fruit preparation reduce antibiotic associated diarrhea and Helicobacter pylori activity

To investigate matrix-specifity of probiotic effects and particularly of the reduction of antibiotics-associated diarrhea, a controlled, randomized, double-blind study was performed, in which 88 Helicobacter pylori-infected but otherwise healthy subjects were given for eight weeks either a) a probiotic fruit yoghurt "mild" containing Lactobacillus acidophilus LA-5 plus Bifidobacterium lactis BB-12, n = 30), b) the same product but pasteurized after fermentation (n = 29) or c) milk acidified with lactic acid (control, n = 29). During week five, a Helicobacter eradication therapy was performed. Helicobacter activity was measured via 13C-2-urea breath tests and antibiotic-associated diarrhoea and other gastrointestinal complaints were recorded by validated questionnaires. In intervention group a, b and c the mean number of days with diarrhoea was 4, 10 and 10 (P<0·05), the frequency of episodes 17%, 7% and 27% (n.s.), and the change in total symptoms score before antibiotics treatment was -1·4 ± 1·1, -1·2 ± 1·1, 2·6 ± 1·1 points/four weeks (P<0·05). All milk products decreased Helicobacter activity by 18 to 45% without significant differences between groups. The observed decrease in Hel. pylori activity seems to be not or not only due to probiotic bacteria but (rather) to components of acidified milk (most probably lactic acid). Fruit-yogurt-like fermented milk products with living probiotic bacteria significantly shorten the duration of antibiotics-associated diarrhoea and improve gastrointestinal complaints. Fruit yogurt-like fermented milk is a matrix suitable for probiotic bacteria.     

Characterizing membrane electrode assemblies for high temperature polymer electrolyte membrane fuel cells using design of experiments

A comparative study of four different high temperature polymer electrolyte membrane fuel cell (HT-PEFC) polybenzimidazole (PBI) based membrane electrode assemblies (MEAs) is undertaken utilizing the design of experiments (DOE) method, a very valuable statistical optimization method, much underutilized in fuel cell research. Single cell voltages are examined as a response (target variable) at two levels (high and low) of four factors (controlled variables); anode and cathode stoichiometry, operating temperature and current density. This yields a two-level, four factor (2⁴) full factorial DOE. The data is used to form a linear regression model for each MEA, which is in turn utilized to predict the cell voltage at random values within the selected ranges of the four factors for validation. The main effects and two factor interactions of each factor are compared to determine their effect on the cell voltage and the underlying physics is examined to determine the best performing MEAs. The PBI based MEA has a much higher tolerance to carbon monoxide (CO) in the fuel stream in comparison with Nafion based MEAs due to the different proton conducting mechanism as well as a higher operating temperature, thus enabling reliable operation of HT-PEFC stacks with reformate containing upto 3% CO.     

Molecular basis of lipase stereoselectivity

Lipases exhibit specific catalytic properties that make them attractive to biotechnological applications. Most important are the broad substrate specificity and the regio‐ and stereoselectivity of lipases. Despite mechanistic and structural similarities lipases differ significantly with respect to stereoselectivity toward natural and synthetic substrates. Models developed to describe and predict stereoselectivity toward certain types of synthetic substrates, e. g., secondary alcohols cannot be applied to natural acylglycerols, that are hydrolyzed by several animal and microbial lipases in a regioselective or stereoselective manner. Therefore, computer‐aided molecular modeling studies were used in order to predict the stereopreference of lipases toward triradylglycerols. Lipase variants with modified stereoselectivity properties toward triacylglycerols were engineered by re‐designing the recombinant enzyme. To understand the interactions governing lipase stereoselectivity towards natural substrates, knowledge of the structure of enzyme‐substrate complexes at the atomic level is essential. Such information can be obtained by X‐ray or NMR analysis of covalent enzyme‐inhibitor complexes. The crystal structures of enzymes complexed with triacylglycerol analog inhibitors allowed the identification of distinct binding sites for the three hydrophobic chains of the inhibitor.     

A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles

Background  

Engineered nanoparticles are becoming increasingly ubiquitous and their toxicological effects on human health, as well as on the ecosystem, have become a concern. Since initial contact with nanoparticles occurs at the epithelium in the lungs (or skin, or eyes), in vitro cell studies with nanoparticles require dose-controlled systems for delivery of nanoparticles to epithelial cells cultured at the air-liquid interface.     

The First Anionic Thia‐Fries Rearrangements at Ferrocene: Ready Access to Trifluoromethylsulfonyl‐Substituted Hydroxyferrocenes and an Extremely High Interannular Stereoinduction between Cyclopentadienyl Ligands

Attempts originally directed towards the generation of ferrocyne (1,2‐dehydroferrocene, 4 ) and ferrocenediyne (1,2,1′,2′‐tetradehydroferrocene, 5 ) by triflate elimination from the respective ferrocenyl triflates led to the discovery of the first anionic thia‐Fries rearrangements at a five‐membered ring. These reactions take place with remarkable efficiency under very mild reaction conditions and yield the respective trifluoromethylsulfonyl‐substituted ferrocenols. Most remarkably, the reaction starting from 1,1′‐ferrocenediyl ditriflate ( 9 ) adopts an extremely high interannular stereoinduction in that exclusively the meso rearrangement product, meso‐2,2′‐bis(trifluoromethylsulfonyl)‐1,1′‐ferrocenediol ( 16 ), is formed, the corresponding racemic product 17 is not observed. It is shown that the second anionic thia‐Fries rearrangement proceeds at a much larger rate than the first one. The stereochemistry and the high rate of this reaction are explained on the basis of electronic as well as steric considerations. The redox behavior of some of the unprecedented ferrocene derivatives has been characterized by cyclovoltammetry.