3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.     

Protective effects of vitamin E against hypercholesterolemia-induced age-related diseases

Hypercholesterolemia is a major risk factor for age-related diseases such as atherosclerosis and Alzheimer’s disease (AD). Changes in human plasma cholesterol levels results from the interaction between multiple genetic and environmental factors. The accumulation of excess cholesterol in blood vessels leads to atherosclerosis. Many studies on this field show that differential expression of oxidative stress-related proteins, lipid metabolism-related enzymes, and receptors response to atherogenic diet. Additionally, excess brain cholesterol has been associated with increased formation and deposition of amyloid-β peptide from amyloid precursor protein which may contribute to the risk and pathogenesis of AD. To consider genetically, more than 50 genes have been reported to influence the risk of late-onset AD. Some of these genes might be also important in cholesterol metabolism and transport. Epidemiological studies have shown an association between high intake and high serum concentrations of antioxidant vitamins like vitamin E and lower rates of ischemic heart diseases. It has been known that vitamin E also inhibits smooth muscle cell proliferation by non-antioxidant mechanism. On the basis of the previous results, vitamin E has been accepted as an important protective factor against hypercholesterolemia-induced age-related diseases.     

Chromatin repair after oxidative stress: Role of PARP-mediated proteasome activation

Oxidative stress is an inevitable process in the nucleus, especially in antitumor chemotherapy, and adaptation by defense mechanisms seems to be one element in the development of long-term resistance to many chemotherapeutic drugs. In this study, a potential chromatin repair mechanism during oxidative stress was investigated in HT22 cells. The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified histone proteins in the nucleus. Poly(ADP-ribosyl)ation reactions also play an important role in DNA repair as a consequence of oxidative damage and single-strand breaks. Such a reaction may occur also with the 20S proteasome—with a known increase in enzymatic activity—and also with histones—reducing their proteolytic susceptibility as shown for the first time here. After hydrogen peroxide treatment of HT22 cells, degradation of the model peptide substrate suc-LLVY-MCA and degradation of oxidized histones by nuclear proteasome increased. During the removal of protein carbonyls, single-strand breaks and 8-hydroxy-2′-deoxyguanosine, proteasome, and poly(ADP-ribose) polymerase-1 enzymes were shown to play tightly interacting roles. Our results following the repair of oxidative damage show the proteolytic activation of proteasome concerning poly(ADP-ribosyl)ation together with a decline in poly(ADP-ribosyl)ation of oxidized histones, leading to a selective recognition of oxidatively modified histones.     

Integration of Metabolic Modeling and Phenotypic Data in Evaluation and Improvement of Ethanol Production Using Respiration-Deficient Mutants of Saccharomyces cerevisiae

Flux balance analysis and phenotypic data were used to provide clues to the relationships between the activities of gene products and the phenotypes resulting from the deletion of genes involved in respiratory function in Saccharomyces cerevisiae. The effect of partial or complete respiratory deficiency on the ethanol production and growth characteristics of hap4Δ/hap4Δ, mig1Δ/mig1Δ, qdr3Δ/qdr3Δ, pdr3Δ/pdr3Δ, qcr7Δ/qcr7Δ, cyt1Δ/cyt1Δ, and rip1Δ/rip1Δ mutants grown in microaerated chemostats was investigated. The study provided additional evidence for the importance of the selection of a physiologically relevant objective function, and it may improve quantitative predictions of exchange fluxes, as well as qualitative estimations of changes in intracellular fluxes. Ethanol production was successfully predicted by flux balance analysis in the case of the qdr3Δ/qdr3Δ mutant, with maximization of ethanol production as the objective function, suggesting an additional role for Qdr3p in respiration. The absence of similar changes in estimated intracellular fluxes in the qcr7Δ/qcr7Δ mutant compared to the rip1Δ/rip1Δ and cyt1Δ/cyt1Δ mutants indicated that the effect of the deletion of this subunit of complex III was somehow compensated for. Analysis of predicted flux distributions indicated self-organization of intracellular fluxes to avoid NAD⁺/NADH imbalance in rip1Δ/rip1Δ and cyt1Δ/cyt1Δ mutants, but not the qcr7Δ/qcr7Δ mutant. The flux through the glycerol efflux channel, Fps1p, was estimated to be zero in all strains under the investigated conditions. This indicates that previous strategies for improving ethanol production, such as the overexpression of the glutamate synthase gene GLT1 in a GDH1 deletion background or deletion of the glycerol efflux channel gene FPS1 and overexpression of GLT1, are unnecessary in a respiration-deficient background.     

Temporary assisting suspension suture technique for successful microvascular anastomosis of extremely small and thin walled vessels for mice transplantation surgery

Dissection and microsurgical anastomosis in small and thin-walled vessels is challenging. Temporary assisting suspension suture technique was developed to overcome those difficulties in establishing successful composite tissue allotransplantation in mice. The operations were performed in 12- to 16-week-old Balb/c mice weighing 25 to 30 grams as both donor and recipient animals. Extended vascularized groin cutaneous flaps based on the superficial epigastric vessels were used. A total of 10 groin cutaneous flaps were transplanted. Three temporary assisting suspension sutures of 11-0 nylon were placed at the 12-, 4-, and 8-o'clock positions to donor and recipient artery and vein before the anastomosis. This technique allowed atraumatic dissection of delicate and thin vessels, prevented vessel wall collapse, and facilitated adequate exposure of the lumen during placement of the permanent microvascular sutures. Thus, the microvascular anastomosis was performed in an unusual manner. The temporary assisting suspension sutures were removed just before the permanent suture was tied down. The mean operation time was 1 hour and 45 minutes with an ischemia time of 1 hour. Ninety-percent success in immediate and late-term patency rates was achieved, which was confirmed by transplant survival. This technique was proven to be useful for microvascular anastomosis in thin-walled vessels and is recommended.     

Comparative Evaluation of Polyphenol Contents and Biological Activities of Five Cistus L. Species Native to Turkey

In this study phytochemical compounds and antioxidant capacity, cytotoxic, antimicrobial and anti-biofilm activities of hydroethanolic extracts of five Cistus species (C. creticus L., C. laurifolius L., C. monspeliensis L., C. parviflorus Lam. and C. salviifolius L.) distributed in Turkey were investigated. (+)-catechin, epigallocatechin gallate, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, luteolin were detected in different amounts. Strongest antioxidant capacities were observed with C. creticus, and C. parvifolius (0.476 and 0.452, respectively). Minimum inhibitory concentrations (MIC) of the extracts were determined between 32 and 128 μg/mL against different bacteria and Candida strains. C. monspeliensis and C. laurifolius extracts were inhibited the biofilm production levels of three Gram-negative bacteria (E. coli, S. enterica, P. aeruginosa), two Gram-positive bacteria (S. aureus, B. subtilis) and three Candida strains (C. albicans, C. parapsilosis, C. krusei). C. creticus extract showed strongest cytotoxic activity against human breast adenocarcinoma (MCF-7) and prostate cell lines (PC-3) (IC₅₀: 14.04±2.78 μg/mL and 34.04±2.74 μg/mL, respectively) among all plants tested.