Increased level of cytokines and matrix metalloproteinases in osteoarthritic subchondral bone

OBJECTIVE: The aim of this study was to investigate the expression of several cytokines, matrix metalloproteinases (MMPs), and tissue inhibitor of matrix metalloproteinases (TIMP)-1 in osteoarthritis (OA) and control sera and different joint tissues. METHODS: Serum, synovial fluid, cartilage, synovial and subchondral bone tissues were examined in OA and control subjects. The protein level of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-8, IL-10 and MMP-2, MMP-3, MMP-9, and TIMP-1 were measured by immunoanalysis. RESULTS: Serum levels of TNF-alpha, MMP-3 and -9 were significantly higher in OA patients than in controls. Conversely, serum IL-10 was decreased in OA patients. CRP was elevated when compared to healthy controls and decreased significantly 6 months after the surgery. In contrast to control samples, OA cartilage and synovium revealed significantly higher MMP-2, -3, -9 and IL-10. IL-1alpha was significantly higher in OA cartilage and IL-8 in OA synovium. Interestingly, MMP-3, -9, TIMP-1 and all tested cytokines were up-regulated in OA subchondral bone. DISCUSSION: This study demonstrates pro-inflammatory condition of OA pathology and supports the idea that vascularized subchondral region may increase the synthesis of cytokines and MMPs leading to degradation of adjacent cartilage.     

Stoichiometry of bacterial anaerobic oxidation of elemental sulfur by ferric iron

The conventional stoichiometry of the oxidation of elemental sulfur by ferric iron in Acidithiobacillus ferrooxidans was not in agreement with our experimental data in terms of ferrous iron and proton formation. Reaction modelling under the actual conditions of bacterial activity resulted in a different stoichiometry, where additional iron species participate in the process to affect the number of released protons. The suggested reaction equation may more accurately predict the intensity of environmental acidification during the anaerobic bioprocess.     

Evaluation of antigenotoxicity effects of umbelliprenin on human peripheral lymphocytes exposed to oxidative stress

The protective properties of a prenylated coumarin, umbelliprenin (UMB), on the human lymphocytes DNA lesions were tested. Lymphocytes were isolated from blood samples taken from healthy volunteers. DNA breaks and resistance to H₂O₂-induced damage were measured using a single-cell microgel electrophoresis technique under alkaline conditions (comet assay). Human lymphocytes were incubated in UMB (10, 25, 50, 100, 200, and 400 μM) alone or a combination of different concentrations of UMB (10, 25, 50, 100, 200, and 400 μM) and 25 μM H₂O₂. Untreated cells, ascorbic acid (AA; 25, 50, 100, 200, and 400 μM) and H₂O₂ (25 μM) were considered as negative control, positive control, and the standard antioxidant agent for our study, respectively. Single cells were analyzed with “TriTek Cometscore version 1.5” software. The DNA damage was expressed as percent tail DNA. UMB exhibited a concentration-dependent increase in protection activity against DNA damage induced by 25 μM H₂O₂ (from 67.28% to 39.17%). The antigenotoxic activity of AA, in the range 0–50 μM, was greater than that of UMB. However, no significant difference (p > 0.05) in the protective activity was found between UMB and AA at concentrations of approximately higher than 50 μM.     

Expression, purification and characterization of soluble human thrombopoietin receptor from Escherichia coli

The extracellular domain (edMpl) of human thrombopoietin (TPO) receptor, c-Mpl was expressed in Escherichia coli by changing some nucleotides before and after the translation initiation codon. The mutations increased the expression by approx. 15-fold. The inclusion bodies were solubilized in 8 M guanidine-HCl under reducing conditions and refolded using a glutathione-redox system. The monomeric form of edMpl was purified to near homogeneity by two successive steps of ion-exchange chromatography using DEAE-Sephacel and Mono Q columns. The purified monomeric edMpl inhibited the TPO-dependent cell proliferation, suggesting that it was binding to TPO. Also, antisera raised against the edMpl bound specifically to the soluble receptor secreted by mammalian cells.     

High concentration of sodium chloride could induce the viable and culturable states of Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis

In the present study, Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis were transferred into Luria–Bertani medium without NaCl (LBWS) and adjusted to various pHs (4, 5, 6 and 7) with lactic acid containing 0·75, 5, 10 and 30% NaCl, and stored at 25°C until the bacterial populations reached below detectable levels on tryptic soy agar (TSA). Although E. coli O157:H7 and S. Enteritidis did not grow on TSA when incubated in LBWS with 30% NaCl for 35 and 7 days, more than 60 and 70% of the bacterial cells were shown to be viable via fluorescent staining with SYTO9 and propidium iodide (PI), respectively, suggesting that a number of cells could be induced into the viable but nonculturable (VBNC) state. These bacteria that were induced into a VBNC state were transferred to a newly prepared tryptic soy broth (TSB) and then incubated at 37°C for several days. After more than 7 days, E. coli O157:H7 and S. Enteritidis regained their culturability. We, therefore, suggest that E. coli O157:H7 and S. Enteritidis entered the VBNC state under the adverse condition of higher salt concentrations and were revived when these conditions were reversed.     

A high density physical map of chromosome 1BL supports evolutionary studies,map-based cloning and sequencing in wheat

Background

As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning.

Results

Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome.

Conclusions

Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing.