Reductive biological treatment of textile effluents

Poster apresentado no "Simpósio Corantes e Pigmentos Orgânicos", na Universidade de Trás-os-Montes e Alto Douro, em Vila Real, Portugal, em Novembro de 2004.Our group is undertaking an investigation on the potential application of ascomycete yeasts to the decolourisation of azo dyes. Two of these strains, Candida zeylanoides (UM2) and Issatchenkia occidentalis (UM41), were isolated from contaminated soil and have been shown to mediate dye decolourisation through reductive cleavage of the azo bond. The rates of colour loss in the presence of yeast cells are independent of their previous exposure to the dye, suggesting that the decolourising activity, under the conditions tested, is constitutive. The process requires intact cells and an external carbon and energy source and depends on pH, temperature and dissolved oxygen. Interestingly, anaerobic conditions do not allow decolourisation. The kinetic study of the cells decolourising activity demonstrated that such activity has a maximum in the late exponential growth phase. Although glucose is the standard carbon and energy source we have also observed decolourisation by cells growing at the expense of ethanol. Decolourisation rates are considerably dependent on the dye structure. Of considerable practical interest is the observation that some of the amines produced by azo dye reduction can be used as carbon and nitrogen sources by the yeast. In order to get further insight on the yeast decolourising activity we have prepared some mutants of a laboratory strain of Saccharomyces cerevisiae and performed inhibition studies. The experimental evidence suggests that major part of the decolourising activity of intact yeast cells is due to a very well characterized plasma membrane redox system.BIOEFTEX Project

Role of the component Fre1p of the plasma membrane ferric reductase on the azo reductase activity of intact Saccharomyces cerevisiae cells

In Press. Aceite em 2005 para publicação na revista Applied and Environmental Microbiology.Unspecific bacterial reduction of azo dyes is a widely studied process in correlation with the biological treatment of coloured waste waters but the enzyme system associated to this bacterial capability has never been positively identified. Several ascomycete yeast strains display similar decolourising activities. The yeast-mediated process requires an alternative carbon and energy source and is independent of previous exposure to the dyes. When substrate dyes are polar their reduction is extracellular, strongly suggesting the involvement of an externally-directed plasma membrane redox system. The present work demonstrates that, in Saccharomyces cerevisiae, the ferric reductase system participates in the extracellular reduction of azo dyes. The Saccharomyces cerevisiae mutant strains Δfre1 and Δfre1Δfre2, but not Δfre2, showed a much reduced decolourising capability, suggesting that, under the conditions tested, Fre1p is a major component of the azo reductase activity.European BIOEFTEX Project

Is Saccharomyces cerevisiae azoreductase the plasma membrane ferric reductase?

Poster apresentado na International Conference on Plasma Membrane Redox Systems and their Role in Biological Stress and Disease, 7, Asilomar, Califórnia, Estados Unidos da América, 14-18 Abril 2004.Unspecific bacterial reduction of azo dyes is a widely studied process in correlation with the biological treatment of coloured waste waters but the enzyme system associated to this bacterial capability has never been positively identified. Several ascomycete yeast strains, isolated by our group from contaminated soils, display similar decolourising activities. A study of the yeast-mediated process in batch culture demonstrated that colour loss was due to a reductase activity, expressed constitutively, which could transform azo dyes into colourless amines. In order to get a better understanding of the azoreductase activity of yeast cells we selected a Saccharomyces cerevisiae strain (BLC0276) with a high decolourising capacity. A likely candidate might be the plasma membrane ferric reductase system. Both azoreductase and ferric reductase have peak activities in the late exponential growth phase, and their substrates -ferricyanide and soluble azo dyes - are impermeant to the cell plasma membrane. This hypothesis was confirmed through two different lines of evidence: the use of different inhibitors and the construction of mutants defective in the iron reductase system. Inhibitors like carbonylcyanide m-chlorophenylhydrazone (CCCP), diphenylene iodonium (DPI), p-chloromercuribenzoate (pCMB) and chloroquine (CQ), were tested. In most cases the percentage inhibition of both activities was similar. The genes Fre1 and Fre2 were deleted and the effect on the decolourising activity was analysed in the mutant strains. The effect of Fre2 deletion was negligible, but fre1 and fre1fre2 strains showed a much reduced decolourising capability. These results demonstrate the involvement of the ferric reductase system in the azo decolourising capacity of S. cerevisiae. However the capacity was not completely removed indicating that cells have an alternative reducing system.BIOEFTEX Project

A contribution for the identification of the azo reductase activity in intact yeast cells

Poster apresentado no International Congress on Yeasts (ICY) -Yeasts in Science and Biotechnology : The Quest for Sustainable Development", 11, Rio de Janeiro, Brasil, Agosto 2004.Azo dyes are synthetic organic colorants which are extensively used in textile, food and cosmetic industries. A large fraction of these dyes is released into the environment even after conventional wastewater treatments. This is a worldwide problem and particularly a problem in regions where textile industries release large quantities of coloured wastewater to water courses. In an attempt to develop a biological treatment for colour removal we isolated several ascomycete yeast strains from contaminated soil based on its capacity to decolourize soluble azo dyes. We studied the process in batch cultures and have demonstrated that the colour loss was due to a reductase activity, expressed constitutively, which could transform azo dyes into colourless amines [1]. In order to understand the process involved in this reduction we selected a Saccharomyces cerevisiae strain (BLC0276) with this capacity. This strain reduced model azo dyes in 8-16 h. We decided to further investigate which enzyme(s) could be involved in this reduction. It was found that in S. cerevisiae there is a membrane redox system with an externally directed reductase – the ferric reductase system, responsible for the extracellular reduction of ferric to ferrous iron previous to uptake, and involving the genes fre1 and fre2. Several know inhibitors of this system were investigated like excess of iron in the medium, CCCP, DPI, chloroquine and p-chloromercurybenzoic acid. Several of these compounds managed to inhibit the decolourizing activity. We also deleted the genes fre1 and fre2 in our strain and analysed the effect on decolourizing activity. Once again the process was retarded in fre1 and fre1fre2 strains. The effect of fre2 deletion was negligible. The deletion of fre1 retarded the decolourizing capability showing the involvement of this system. However the capacity was not completely removed indicating that cells have an alternative reducing system. [1] Ramalho, P.A., Scholze, H., Cardoso, M.H., Ramalho, M.T., Oliveira-Campos, A.M. 2002 Improved conditions for the aerobic reductive decolourization of azo dyes by Candida zeylanoides. Enz Microbiol Technol 31: 848-854.BIOEFTEX Project

Endothelial progenitor cells in diabetic patients with myocardial infarction – Can statins improve their function?

The effect of statins on endothelial progenitor cells (EPCs) function derived from diabetic patients (DMpts) with acute myocardial infarction (AMI) is unknown. In this study we assess the response of early and late EPCs from diabetic versus non-diabetic patients (NDMpts) with AMI to statins. EPCs were obtained from 10 diabetic and 10 age-matched non-diabetic male patients with AMI. For each patient, cultures of early and late EPCs were performed under 4 different conditions: normal glucose concentration (control); high glucose concentration; normal glucose concentration with atorvastatin supplementation and normal glucose concentration with pravastatin supplementation. To compare the effect of these treatments on EPC function in DMpts versus NDMpts, we performed in vitro: EPC colony-forming units (CFU) assay; cell cycle analysis; viability assessment and expression of the surface markers CXCR4, CD133, CD34 and KDR. Under control conditions, CFU numbers were reduced in DMpts-derived EPCs when compared to those of NDMpts (1.4±0.8 vs 2.6±1.2 CFU/well, P=0.021). When early EPCs from DMpts were cultured in the presence of statins, CFU capacity was restored, surmounting that of NDMpts under control conditions. Statins significantly improved viability of early EPCs and delayed the onset of late EPCs senescence, even in cells from DMpts. In addition, statins induced approximately a 2-fold increase in the proportion of late EPCs in S-phase of the cell cycle (P<0.05). Statins have a beneficial effect on both early and late EPCs from DMpts with AMI. Despite the functional impairment of EPCs from DMpts, they exhibit similar responsiveness to statins as equivalent cells from NDMpts

Mesenchymal stem/stromal cells : disrupting cell therapy storage and distribution with hypothermic preservation of adipose-derived mesenchymal stromal cells

Background & Aim: Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily burdened process. Encapsulation at hypothermic temperatures (e.g.,2–8°C) could be a feasible alternative. In this study, we aim to determine the ability of alginate encapsulation to maintain cell viability, identity, and function in the context of MSC-based therapy manufacturing. Methods, Results & Conclusion: Adipose tissue-derived mesenchymal stromal cells (MSC(AT)) expanded using fetal bovine serum (FBS)- (MSC-FBS) or human platelet lysate (HPL)-supplemented mediums (MSC-HPL) were encapsulated in alginate beads (BeadReady™ kits kindly provided by Atelerix) for 30 min, 5 days, and 12 days. After bead release, cell recovery and viability were determined to assess encapsulation performance. MSC identity and functional immunophenotype, MSC tri-lineage differentiation potential, metabolic activity, and hematopoietic support capacity were determined and compared between timepoints. MSC(AT) were able to survive encapsulated for a standard transportation period of 5 days, with recovery values of 56 ± 5% for MSC-FBS and 77 ± 6% for MSC-HPL (which is a negligible drop compared to earlier timepoints). Importantly, MSC function did not suffer from encapsulation, with recovered cells showing robust differentiation potential, expression of immunomodulatory molecules, and hematopoietic support capacity. MSC(AT) encapsulation was proven possible for a remarkable 12 day period. There is currently no solution to completely replace cryopreservation in CGT logistics and supply chain, although encapsulation has shown potential to act as a serious competitor.info:eu-repo/semantics/publishedVersio

Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders

Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes