Reflux induces DNA strand breaks and expression changes of MMP1+9+14 in a human miniorgan culture model

Gastroesophageal reflux disease has been implicated in the pathogenesis of adenocarcinoma of the oesophagus. The same applies to laryngopharyngeal reflux (LPR) and squamous cell cancer of the head and neck, but so far, this link has not been proven. The impact of low pH and bile acids has not been studied extensively in cells other than oesophageal cancer cell lines and tissue. The aims of this study were to investigate the pathogenic potential of reflux and its single components on the mucosa of the upper respiratory tract. We measured DNA stability in human miniorgan cultures (MOCs) and primary epithelial cell cultures (EpCs) in response to reflux by the alkaline comet assay. As matrix metalloproteinases (MMPs) are involved in extracellular matrix remodelling processes and may contribute to cancer progression, we studied the expression of MMP1, -9, and -14 in MOCs, EpC, UM-SCC-22B, and FADU_DD. DNA strand breaks (DNA-SBs) increased significantly at low pH and after incubation with human or artificial gastric juice. Single incubation with glycochenodeoxycholic acid also showed a significant increase in DNA-SBs. In epithelial cell cultures, human gastric juice increased the number of DNA-SBs at pH 4.5 and 5.5. Artificial gastric juice significantly up regulated the gene expression of MMP9. Western blot analysis confirmed the results of gene expression analysis, but the up regulation of MMP1, -9, and -14 was donor-specific. Reflux has the ability to promote genomic instability and may contribute to micro environmental changes suitable for the initiation of malignancy. Further functional gene analysis may elucidate the role of laryngopharyngeal reflux in the development of head neck squamous cell carcinoma (HNSCC).     

A modified metabolic model for mixed culture fermentation with energy conserving electron bifurcation reaction and metabolite transport energy

A modified metabolic model for mixed culture fermentation (MCF) is proposed with the consideration of an energy conserving electron bifurcation reaction and the transport energy of metabolites. The production of H₂ related to NADH/NAD⁺ and Fdred/Fdox is proposed to be divided in three processes in view of energy conserving electron bifurcation reaction. This assumption could fine‐tune the intracellular redox balance and regulate the distribution of metabolites. With respect to metabolite transport energy, the proton motive force is considered to be constant, while the transport rate coefficient is proposed to be proportional to the octanol–water partition coefficient. The modeling results for a glucose fermentation in a continuous stirred tank reactor show that the metabolite distribution is consistent with the literature: (1) acetate, butyrate, and ethanol are main products at acidic pH, while the production shifts to acetate and propionate at neutral and alkali pH; (2) the main products acetate, ethanol, and butyrate shift to ethanol at higher glucose concentration; (3) the changes for acetate and butyrate are following an increasing hydrogen partial pressure. The findings demonstrate that our modified model is more realistic than previous proposed model concepts. It also indicates that inclusion of an energy conserving electron bifurcation reaction and metabolite transport energy for MCF is sound in the viewpoint of biochemistry and physiology. Biotechnol. Bioeng. 2013; 110: 1884–1894. © 2013 Wiley Periodicals, Inc.     

Activin A regulates growth of gastro‐intestinal epithelial cells by mediating epithelial‐mesenchymal interaction

The importance of epithelial–mesenchymal interaction on the development of gastro‐intestinal (GI) organs has been repeatedly reported, but its molecular mechanism has not been fully understood though several factors including hepatocyte growth factor and endothelin‐3 have been shown to mediate it. Activins have been demonstrated to play important roles in the regulation of organogenesis in vertebrates, but their roles in the regulation of growth and differentiation of GI organs remain to be solved. In the present study, we examined expression of activins in developing rat GI tract, and found that inhibin bA encoding activin A was specifically expressed by GI mesenchymes, while inhibin bB encoding activin B was expressed by both epithelial and mesenchymal components. We then examined the effect of activin A on the growth of fetal rat GI epithelial cells in primary culture. We found that activin A inhibited the growth of forestomach and glandular stomach epithelial cells while it stimulated the growth of colonic epithelial cells. These results suggest that activin A secreted from GI mesenchymes region‐specifically regulates the growth of attaching epithelial cells. We thus conclude that activin A mediates epithelial‐mesenchymal interaction in the developing GI tract.     

Potato purple top phytoplasma‐induced disruption of gibberellin homeostasis in tomato plants

Phytoplasmas are phloem‐inhabiting, cell wall‐less bacteria that cause numerous plant diseases worldwide. Plants infected by phytoplasmas often exhibit various symptoms indicative of hormonal imbalance. In this study, we investigated the effects of potato purple top (PPT) phytoplasma infection on gibberellin homeostasis in tomato plants. We found that PPT phytoplasma infection caused a significant reduction in endogenous levels of gibberellic acid (GA₃). The decrease in GA₃ content in diseased plants was correlated with down regulation of genes responsible for biosynthesis of bioactive GAs ( GA20ox1 and GA3ox1) and genes involved in formation of GA precursors [geranyl diphosphate synthase (GPS) and copalyldiphosphate synthase (CPS)]. Exogenous application of GA₃ at 200 µmol L^?1 was able to restore the GA content in infected plants to levels comparable to those in healthy controls, and to attenuate the characteristic ‘big bud’ symptoms induced by the phytoplasma. The interesting observation that PPT phytoplasma‐infected plants had prolonged low expression of key GA biosynthesis genes GA20ox1 and GA3ox1 under GA deficiency conditions led us to hypothesise that there was a diminished sensitivity of the GA metabolism feedback regulation, especially GA biosynthesis negative feedback regulation, in those affected plants, and such diminished sensitization in early stages of infection may represent a central element of the phytoplasma‐induced disruption of GA homeostasis and pathogenesis.     

An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture

Oligosaccharides attached to Asn297 in each of the C_H2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.     

Susceptibility of tall fescue to Rhizoctonia zeae infection as affected by endophyte symbiosis

Endophytic fungi belonging to the genus Neotyphodium often form symbiotic associations with grasses. The host plants usually benefit from the association with an endophyte. Presence of the symbiont may increase host resistance to infection by some pathogens. However, the exact mechanism of the lower susceptibility of endophyte‐infected plants to diseases is still unclear. Growth chamber trials were conducted to determine whether (a) tall fescue plants infected with the endophyte Neotyphodium coenophialum (E+) are more resistant to sheath and leaf spot disease caused by Rhizoctonia zeae than endophyte‐free (E?) plants, and (b) R. zeae growth inhibition is associated with endophyte presence. Tall fescue genotypes, each symbiotic with a genetically different native endophyte strain, were inoculated with isolates of R. zeae. The tillers infection by R. zeae, density of endophyte hyphae and content of total phenolic compounds in tillers were studied. Antifungal activity of the N. coenophialum towards R. zeae, Rhizoctonia solani, Bipolaris sorokiniana and Curvularia lunata was also investigated in dual‐culture assays. For Tf3, Tf4, TfA2 and TfA9 tall fescue genotypes, the E+ plants had reduced R. zeae infection. In the Tf9 and Tf8085 genotypes, R. zeae infection was similar for both E+ and E? plants. The strongest effect was observed for the Tf4 endophyte. A strongly positive correlation (r = 0.94) occurred between endophyte hyphal density and disease index across all tall fescue genotypes. Dual‐culture assays showed no inhibitory interaction between the seven endophyte strains and the R. zeae isolates; however, some endophytes inhibited R. solani, B. sorokiniana and C. lunata. Endophyte presence increased the production of phenolic compounds by the host grasses. The level of phenolics also differed significantly depending on the time of analysis after inoculation of plants by R. zeae. The results indicate that N. coenophialum can suppress disease severity caused by R. zeae infection. The mechanism of higher resistance of E+ plants is likely not based on direct inhibition such as antibiosis or competition. Thus, the induction of specific mechanisms in the host plant, for example, production of phenolic compounds, seems to be the main way of providing resistance to the grass by the endophyte.     

Generation of robust vascular networks from cardiovascular blast populations derived from human induced pluripotent stem cells in vivo and ex vivo organ culture system

Vascular network formation is a key therapeutic event in regenerative medicine because it is essential for mitigating or ameliorating ischemic conditions implicated in various diseases and repair of tissues and organs. In this study, we induced human induced pluripotent stem cells (hiPSCs) to differentiate into heterogeneous cell populations which have abilities to form vascular vessel-like structures by recapitulating the embryonic process of vasculogenesis in vitro. These cell populations, named cardiovascular blast populations (CBPs) in this report, primarily consisted of CD31⁺ and CD90⁺ cells.     

Reactive black-5 azo dye treatment in suspended and attach growth sequencing batch bioreactor using different co-substrates

Treatment of textile wastewater is a big challenge because of diverse chemical composition, high chemical strength and color of the wastewater. In the present study, treatment of wastewater containing reactive black-5 azo dye was studied in anaerobic sequencing batch bioreactor (SBBR) using mixed liquor suspended solids (MLSS) from suspended and attach growth bioreactors. MLSS at concentration of 1000 mg/L and reactive black-5 azo dye at 100 mg/L were used. A culture (10⁸–10⁹ CFU/ml) of pre-isolated bacterial strains (Psychrobacter alimentarius KS23 and Staphylococcus equorum KS26)) capable of degrading azo dyes in mineral salt medium was used to accelerate the treatment process in bioreactor. Different combinations of sludge, culture and dye were used for treatment using different co-substrates. About 85% COD removal was achieved by consortium (MLSS + KS23 + KS26) after 24 h in attach growth bioreactor. Similarly, 92% color removal was observed with consortium in attach growth bioreactor compared to 85% color removal in suspended bioreactor. Addition of bacterial culture (20%, v/v) to the bioreactor could enhance the rate of color removal. This study suggests that biotreatment of wastewater containing textile dyes can be achieved more efficiently in the attach growth bioreactor using yeast extract as a co-substrate and MLSS augmented with dye-degrading bacterial strains.     

On the conspecificity of marine and freshwater Bangia in Britain

The salinity responses of marine and freshwater Bangia have been studied in laboratory culture. Although both isolates possessed salinity tolerances which were dependent upon the salinity regimes of their original habitats, neither was restricted in its salinity tolerances to that salinity range alone. The marine isolate was able to proliferate after a single step transfer into a freshwater-based medium. Certain cells within the thallus appeared to be resistant to the sudden decrease in salinity and these developed into dwarf plantlets, following a period of dormancy. An hypothesis is proposed that Bangia occurring in freshwater is a well-adapted ecotype of the marine form, thus supporting the conspecific theory.     

Direct Regulation of Na+-Dependent myo-Inositol Transport by Sugars in Retinal Pigment Epithelium: Role of Phorbol Ester and Staurosporin

An Na⁺-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32). The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells. RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, α-methyl-D-glucoside (αMG); 1–5 mM nonradioactive MI, pyruvate, or lactate; or 0.2–20 µM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks. The capacity of RPE cells to accumulate ³H-MI (ratios of intracellular transported radioactive MI, [MI]_i, to external free MI concentration, [MI]_i/[MI]₀) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM αMG, respectively, compared to cells grown in basic growth media. The rate of uptake of ³H-MI also was reduced to 63 ± 15% or 48 ± 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively. In addition, cellular capacity to bind to [³H]phlorizin was reduced to 52 ± 7%, 61 ± 5%, or 38 ± 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM αMG, or 5 mM nonradioactive MI, respectively. Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI. An 18 ± 8% reduction in [³H]thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12–14 days, compared to cells grown with 5 mM glucose. Chronic treatment (12–14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, [³H]phlorizin binding, cell number, and DNA synthesis. However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells. Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake. In contrast to its chronic effect, a 60-min incubation (acute effect) of cells in the presence of TPA, with or without inclusion of stauropsorin, did not alter the uptake of ³H-MI into RPE cells, regardless of glucose levels in the growth media. These studies indicated that glucose itself, and not glucose metabolites, regulated uptake of MI into primary cultures of RPE cells. In addition, glucose-induced down-regulation of MI uptake was not mediated through the protein kinase C pathway, but the staurosporin-inhibited, TPA-stimulated protein kinase C was partly responsible for growth and proliferation of RPE cells.