Osteoid-mimicking dense collagen/chitosan hybrid gels

Bone extracellular matrix (ECM) is a 3D network, composed of collagen type I and a number of other macromolecules, including glycosaminoglycans (GAGs), which stimulate signaling pathways that regulate osteoblast growth and differentiation. To model the ECM of bone for tissue regenerative approaches, dense collagen/chitosan (Coll/CTS) hybrid hydrogels were developed using different proportions of CTS to mimic GAG components of the ECM. MC3T3-E1 mouse calvaria preosteoblasts were seeded within plastically compressed Coll/CTS hydrogels with solid content approaching that of native bone osteoid. Dense, cellular Coll/CTS hybrids were maintained for up to 8 weeks under either basal or osteogenic conditions. Higher CTS content significantly increased gel resistance to collagenase degradation. The incorporation of CTS to collagen gels decreased the apparent tensile modulus from 1.82 to 0.33 MPa. In contrast, the compressive modulus of Coll/CTS hybrids increased in direct proportion to CTS content exhibiting an increase from 23.50 to 55.25 kPa. CTS incorporation also led to an increase in scaffold resistance to cell-induced contraction. MC3T3-E1 viability, proliferation, and matrix remodeling capability (via matrix metalloproteinase expression) were maintained. Alkaline phosphatase activity was increased up to two-fold, and quantification of phosphate mineral deposition was significantly increased with CTS incorporation. Thus, dense Coll/CTS scaffolds provide osteoid-like models for the study of osteoblast differentiation and bone tissue engineering.     

A SUMO-Dependent Step during Establishment of Sister Chromatid Cohesion

Cohesin is a protein complex that ties sister DNA molecules from the time of DNA replication until the metaphase to anaphase transition. Current models propose that the association of the Smc1, Smc3, and Scc1/Mcd1 subunits creates a ring-shaped structure that entraps the two sister DNAs [1]. Cohesin is essential for correct chromosome segregation and recombinational repair. Its activity is therefore controlled by several posttranslational modifications, including acetylation, phosphorylation, sumoylation, and site-specific proteolysis. Here we show that cohesin sumoylation occurs at the time of cohesion establishment, after cohesin loading and ATP binding, and independently from Eco1-mediated cohesin acetylation. In order to test the functional relevance of cohesin sumoylation, we have developed a novel approach in budding yeast to deplete SUMO from all subunits in the cohesin complex, based on fusion of the Scc1 subunit to a SUMO peptidase Ulp domain (UD). Downregulation of cohesin sumoylation is lethal, and the Scc1-UD chimeras have a failure in sister chromatid cohesion. Strikingly, the unsumoylated cohesin rings are acetylated. Our findings indicate that SUMO is a novel molecular determinant for the establishment of sister chromatid cohesion, and we propose that SUMO is required for the entrapment of sister chromatids during the acetylation-mediated closure of the cohesin ring.     

Altitudinal variation of demographic life-history traits does not mimic latitudinal variation in natterjack toads (Bufo calamita)

In anuran amphibians, age- and size-related life-history traits vary along latitudinal and altiudinal gradients. In the present study, we tested the hypothesis that altitudinal and latitudinal effects cause similar responses by assessing demographic life-history traits in nine Bufo calamita populations inhabiting elevations from sea level to 2270 m. Skeletochronologically determined age at maturity and longevity increased at elevations exceeding 2000 m, but female potential reproductive lifespan (PRLS) did not increase with altitude, as it did with latitude. Integrating the available evidence, it was found that lifetime fecundity of natterjacks decreased at the upper altitudinal range because PRLS was about the same as in lowland populations but females were smaller. In contrast, small size of northern females was compensated for by increased PRLS which minimised latitudinal variation of lifetime fecundity. Thus, this study provides evidence that altitudinal effects on life-history traits do not mimic latitudinal effects. Life-history trait variation along the altitudinal gradient seems to respond directly to the shortening of the annual activity period. As there is no evidence for increasing mortality in highland populations, reduced lifetime fecundity may be the ultimate reason for the natterjacks' inability to colonise elevations exceeding 2500 m.     

Indene-based frameworks targeting the 5-HT6 serotonin receptor: Ring constraint in indenylsulfonamides using cyclic amines and structurally abbreviated counterparts

Further studies in quest of 5-HT₆ serotonin receptor ligands led to the design and synthesis of a few selected examples of N-(inden-5-yl)sulfonamides with a ring-constrained aminoethyl side chain at the indene 3-position, some of which exhibited a high binding affinity, such as the pyrrolidine analogue 28 (K_i = 3 nM). Moreover, the structurally abbreviated N-(inden-5-yl)sulfonamides showed K_i values ?43 nM, which indicates that neither the N,N-aminoethyl nor the conformationally restricted aminoethyl side arm at the indene 3-position are required for binding. Selected compounds were then tested in a functional cAMP stimulation assay and found to act as 5-HT₆ antagonists, although with moderate potency at the micromolar level.     

Quantitative PCR Monitoring of Antibiotic Resistance Genes and Bacterial Pathogens in Three European Artificial Groundwater Recharge Systems

Aquifer recharge presents advantages for integrated water management in the anthropic cycle, namely, advanced treatment of reclaimed water and additional dilution of pollutants due to mixing with natural groundwater. Nevertheless, this practice represents a health and environmental hazard because of the presence of pathogenic microorganisms and chemical contaminants. To assess the quality of water extracted from recharged aquifers, the groundwater recharge systems in Torreele, Belgium, Sabadell, Spain, and Nardò, Italy, were investigated for fecal-contamination indicators, bacterial pathogens, and antibiotic resistance genes over the period of 1 year. Real-time quantitative PCR assays for Helicobacter pylori, Yersinia enterocolitica, and Mycobacterium avium subsp. paratuberculosis, human pathogens with long-time survival capacity in water, and for the resistance genes ermB, mecA, blaSHV-5, ampC, tetO, and vanA were adapted or developed for water samples differing in pollutant content. The resistance genes and pathogen concentrations were determined at five or six sampling points for each recharge system. In drinking and irrigation water, none of the pathogens were detected. tetO and ermB were found frequently in reclaimed water from Sabadell and Nardò. mecA was detected only once in reclaimed water from Sabadell. The three aquifer recharge systems demonstrated different capacities for removal of fecal contaminators and antibiotic resistance genes. Ultrafiltration and reverse osmosis in the Torreele plant proved to be very efficient barriers for the elimination of both contaminant types, whereas aquifer passage followed by UV treatment and chlorination at Sabadell and the fractured and permeable aquifer at Nardò posed only partial barriers for bacterial contaminants.     

A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein–Raf1 and the Raichu-KRas probe, we identified for the first time in vivo–active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14–MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.     

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Thailand

Tissue banking started in Thailand in 1979. Five years after this, the Bangkok Biomaterial Centre (BBC) was established in the Faculty of Medicine, Siriraj Hospital, with the support of the IAEA program. The objective of the Centre was to provide sterile bones and tissues for clinical use. Through the passage of time, the Bangkok Biomaterial Centre has gained confidence from the end user and by 2007 has processed 33,872 allografts from 491 deceased donors and 4,035 live donors were used in medical treatment in 3,596 patients in more than 79 different hospitals. More than 305 surgeons from Thailand used the tissue produced in the BBC. At the beginning of its work the BBC concentrate its activities on the production of the following tissues: freeze dried bone, freeze dried dura mater and freeze dried fascia lata. All of these tissues were sterilised using ethylene oxide gas until the end of year 1984. Since 1985 the BBC sterilise tissue using ionising radiation. The BBC is now producing deep-frozen; bone tendon, cartilage, trachea and soft tissue; freeze-dried; bone, fascia lata, dura mater, amniotic membrane, bone hydroxyapatite, bone tablet and fresh preserved amniotic membrane Yongyudh Vajaradul is a Founder of Bangkok Biomaterial Centre and also a President of TATB, Bangkok, Thailand. Jorge Morales Pedraza is a former IAEA Interregional Project Manager, Vienna, Austria.