Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.     

Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities

Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones.     

A barcode analysis of the genus Lobophora (Dictyotales,Phaeophyceae) in the western Atlantic Ocean with four novel species and the epitypification of L. variegata (J.V. Lamouroux) E.C. Oliveira

In the western Atlantic Ocean, the brown algal genus Lobophora is currently represented by a single species, L. variegata, with a type locality designated by Lamouroux as ‘Antilles’. In this study, we used molecular-assisted alpha taxonomy (MAAT) to assess species diversity of Lobophora in Bermuda, the Florida Keys, St. Croix and Guadeloupe (Lesser Antilles). Using cox1 and cox3 sequences as barcode markers, five species of Lobophora, four of them novel, were delineated, all previously having been identified in the area as L. variegata. Our morphological and habitat studies, made possible by abundant sampling, have revealed unique characters for each of these western Atlantic species, including distinct cellular arrangements, as well as different depth ranges for certain species. Observations made from Lamouroux’s holotype of Dictyota variegata (= Lobophora variegata) allowed us to assess the anatomy of this species, which enabled us to easily align this early taxon to one of our genetic species from the western Atlantic. As the type was unavailable for genetic analysis, we selected a recent St. Croix (Virgin Is., Antilles) specimen as the epitype to support it with molecular sequence data.     

Anthocyanins Double the Shelf Life of Tomatoes by Delaying Overripening and Reducing Susceptibility to Gray Mold

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Multiple myeloma and occupation: A pooled analysis by the International Multiple Myeloma Consortium

Objective: We investigated occupational risk of multiple myeloma (MM) in a pooled analysis of five international case–control studies. Methods: We calculated the odds ratio and its 95% confidence interval for selected occupations with unconditional regression analysis in 1959 MM cases and 6192 controls, by pooling study-specific risks using random-effects meta-analysis. Exposure to organic solvents was assessed with a job-exposure matrix (JEM). Results: Gardeners and nursery workers combined, most likely exposed to pesticides, showed a 50% increase in risk (OR = 1.50, 95% CI 0.9–2.3), while other farming jobs did not. Metal processors (OR = 1.55, 95% CI 0.9–2.3), female cleaners (OR = 1.32, 95% CI 1.0–1.8), and high level exposure to organic solvents (OR = 1.38, 95% CI 0.96–1.8) also showed moderately increased risks. Conclusions: Additional case–control studies of MM aetiology are warranted to further investigate the nature of the repeatedly reported increase in MM risk in several occupational groups.     

Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane

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Continuous Solvent/Detergent Virus Inactivation Using a Packed‐Bed Reactor

Continuous virus inactivation (VI) remains one of the missing pieces while the biopharma industry moves toward continuous manufacturing. The challenges of adapting VI to the continuous operation are two‐fold: 1) achieving fluid homogeneity and 2) a narrow residence time distribution (RTD) for fluid incubation. To address these challenges, a dynamic active in‐line mixer and a packed‐bed continuous virus inactivation reactor (CVIR) are implemented, which act as a narrow RTD incubation chamber. The developed concept is applied using solvent/detergent (S/D) treatment for inactivation of two commonly used model viruses. The in‐line mixer is characterized and enables mixing of the viscous S/D chemicals to ±1.0% of the target concentration in a small dead volume. The reactor's RTD is characterized and additional control experiments confirm that the VI is due to the S/D action and not induced by system components. The CVIR setup achieves steady state rapidly before two reactor volumes and the logarithmic reduction values of the continuous inactivation process are identical to those obtained by the traditional batch operation. The packed‐bed reactor for continuous VI unites fully continuous processing with very low‐pressure drop and scalability.     

cGMP-independent inhibition of integrin alphaIIbbeta3-mediated platelet adhesion and outside-in signalling by nitric oxide

We examined the influence of S-nitrosoglutathione (GSNO) on alpha(IIb)beta(3) integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin beta(3) phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of beta(3) on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin beta(3) phosphorylation.