Production of mexican brown macroalgae fucoidan and fucosidases under an integral green technology bioproceses by the biorefinery concept

Marine ecosystem can be considered a rather exploited source of natural substances with enormous bioactive potential. In Mexico macro-algae study remain forgotten for research and economic purposes besides the high amount of this resource along the west and east coast. For that reason the Bioferinery Group of the Autonomous University of Coahuila, have been studying the biorefinery concept in order to recover high value byproducts of Mexican brown macro-algae including polysaccharides and enzymes to be applied in food, pharmaceutical and energy industry. Brown macroalgae are an important source of fucoidan, alginate and laminarin which comprise a complex group of macromolecules with a wide range of important biological properties such as anticoagulant, antioxidant, antitumoral and antiviral and also as rich source of fermentable sugars for enzymes production. Additionally, specific enzymes able to degrade algae matrix (fucosidases, sulfatases, aliginases, etc) are important tools to establish structural characteristics and biological functions of these polysaccharides. The aims of the present work were the integral study of bioprocess for macroalgae biomass exploitation by the use of green technologies as hydrothermal extraction and solid state fermentation in order to produce polysaccharides and enzymes (fucoidan and fucoidan hydrolytic enzymes). This work comprises the use of the different bioprocess phases in order to produce high value products with lower time and wastes.

Aplicação de celulases e xilanases na reciclagem de fibras de papel

Tese de Doutoramento em Engenharia Química e Biológica (área de conhecimento em Engenharia Enzimática e das Fermentações)

Producción in vitro de Embriones Bovinos Transgénicos mediante Inyección Intracitoplasmática de Espermatozoides (ICSI) con Enzimas de Restricción. In vitro Production of Transgenic Bovine Embryos using Intracitoplasmic Sperm Injection (ICSI) and Restriction Enzymes

La ingeniería genética y la reprogramación de organismos vivos representan las nuevas fronteras biotecnológicas que permitirán generar animales con modificaciones precisas en sus genomas para un sinnúmero de aplicaciones biomédicas y agropecuarias. Las técnicas para inducir modificaciones génicas intencionales en animales, especialmente en especies mayores de interés agropecuario, se encuentran rezagadas si se compara con los avances significativos que se han producido en el área de la transgénesis de roedores de laboratorio, especialmente el ratón. Es así que, el presente proyecto persigue desarrollar y optimizar protocolos para generar embriones bovinos transgénicos para aplicaciones biotecnológicas. La estrategia propuesta, se basa en conseguir la presencia simultánea en el interior celular de una enzima de restricción (I-SceI) más un transgén (formado por casetes de expresión de una proteína fluorescente -ZsGreen1- y neomicina fosfotransferasa). Específicamente, proyectamos estudiar una vía alternativa para generar embriones bovinos transgénicos mediante la incorporación del transgén (casetes ZsGreen1 y neo) flanqueado por sitios I-SceI más la enzima I-SceI al interior del ovocito junto con el espermatozoide durante la técnica conocida como inyección intracitoplasmática de espermatozoides (ICSI). Los embriones así generados se cultivarán in vitro, inspeccionándolos diariamente para detectar la emisión de fluorescencia, indicativa de la expresión de la proteína ZsGreen1. Los embriones que alcancen el estado de blastocisto y expresen el transgén se transferirán quirúrgicamente al útero de ovejas sincronizadas y se mantendrán durante 7 días. Al cabo de este período, los embriones se recolectarán quirúrgicamente del útero ovino y se transportarán al laboratorio para determinar el número de sitios de integración y número de copias del transgén mediante el análisis de su ADN por Southern blot. Se prevé que los resultados de esta investigación permitirán sentar las bases para el desarrollo de métodos eficientes para obtener modificaciones precisas en el genoma de los animales domésticos para futuras aplicaciones biotecnológicas. Genetic engineering and reprogrammed organisms represent the new biotechnological frontiers which will make possible to generate animals with precise genetic modifications for agricultural and biomedical applications. Current methods used to generate genetically modified large animals, lay behind those used in laboratory animals, specially the mouse. Therefore, we seek to develop and optimize protocols to produce transgenic bovine embryos through the use of a non-viral vector. The strategy involves the simultaneous presence inside the cell of a restriction enzyme (I-SceI) and a transgene (carrying cassettes for a fluorescent protein -ZsGreen1- and neomycin phosphotransferase) flanked by restriction sites for the endonuclease. We plan to develop an alternative approach to generate transgenic bovine embryos by coinjecting the transgene flanked by I-SceI restriction sites plus the enzyme I-SceI along with the spermatozoon during the technique known as intracytoplasmic sperm injection (ICSI). Embryos will be cultured in vitro and inspected daily with a fluorescence microscope to characterize transgene expression. Embryos that reach the blastocyst stage and express the transgene will be surgically transfer to the uterus of a synchronized ewe. After 7 days, the embryos will be flushed out the ovine uterus and transported to the laboratory to determine the number of integration sites and transgene copies by Southern blot. We anticipate that results from this research will set the stage for the development of efficient strategies to achieve precise genetic modifications in large domestic animals for future biotechnological applications.

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

Peroxisomal polyhydroxyalkanoate biosynthesis is a promising strategy for bioplastic production in high biomass crops.

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers with diverse plastic-like properties. PHA biosynthesis in transgenic plants is being developed as a way to reduce the cost and increase the sustainability of industrial PHA production. The homopolymer polyhydroxybutyrate (PHB) is the simplest form of these biodegradable polyesters. Plant peroxisomes contain the substrate molecules and necessary reducing power for PHB biosynthesis, but peroxisomal PHB production has not been explored in whole soil-grown transgenic plants to date. We generated transgenic sugarcane (Saccharum sp.) with the three-enzyme Ralstonia eutropha PHA biosynthetic pathway targeted to peroxisomes. We also introduced the pathway into Arabidopsis thaliana, as a model system for studying and manipulating peroxisomal PHB production. PHB, at levels up to 1.6%-1.8% dry weight, accumulated in sugarcane leaves and A. thaliana seedlings, respectively. In sugarcane, PHB accumulated throughout most leaf cell types in both peroxisomes and vacuoles. A small percentage of total polymer was also identified as the copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in both plant species. No obvious deleterious effect was observed on plant growth because of peroxisomal PHA biosynthesis at these levels. This study highlights how using peroxisomal metabolism for PHA biosynthesis could significantly contribute to reaching commercial production levels of PHAs in crop plants.

Comparison of a multiplexed bead-based assay with an immunofluorescence and an enzyme-immuno assay for the assessment of Epstein-Barr virus serologic status

Dans cette étude, nous avons testé la performance diagnostique d'une nouvelle technique d'analyse multiplexée qui permet la détection d'anticorps de différentes spécificités dans la même réaction. En l'absence de gold standard, nous avons choisi de comparer la performance diagnostique de l'analyse avec deux méthodes de référance que sont l'IF et EIA, et avec un consensus déterminé selon une règle de majorité entre les trois méthodes.¦393 sérums analysés par IF, conservés par congélation, ont été décongelés pour être analysés par EIA et BBA. Pour chaque sérum, les anticorps recherchés ont été les anti-VCA (Viral Capsid Antigen) IgM, anti-VCA IgG et anti-EBNA (Epstein-Barr Nuclear Associated) IgG. Les échantillons ont été classés en cinq groupes selon les résultats de l'IF : séronégatifs, infections aiguës, infections anciennes et deux types d'indéterminés.¦Pour chaque méthode, le résultat numérique (index ou titre) des analyses est converti en termes de positif, négatif ou douteux. Pour le résultat de chaque type d'anticorps, un consensus est établi selon une règle de majorité entre les trois méthodes, permettant une interprétation du stade de l'infection. Puis l'interprétation de chacune des méthodes a été comparée au consensus. Nous avons également comparé les trois méthodes les unes aux autres concernant la détection des anticorps.¦Globalement, nous observons une bonne corrélation qualitative entre les trois approches pour détecter les anti-VCA IgG et IgM. Pour pour les anti-EBNA IgG, il y a une divergence notable entre l'IF et les deux autres méthodes, l'IF apparaissant moins sensible que les autres méthodes, résultant en un nombre accru d'interprétations indéterminées du stade de l'infection.¦L'origine de cette divergence ne peut être due à une perte d'anticorps liée au stockage de longue durée des échantillons. En effet, EIA et BBA restent plus sensibles que IF, dont l'analyse a été faite sur des sérums frais.¦Cette divergence ne semble pas non plus être due aux différents antigènes utilisés par les trois méthodes. EIA et BBA utilisent le même antigène recombinant EBNA-1, alors que l'IF utilise des "cellules lymphoïdes choisies pour leur production sélective d'antigènes EBNA". Ces cellules sont probablement des cellules infectées par EBV qui devraient exprimer plus d'antigènes de latence que seul EBNA-1. Cette différence devrait donc plutôt en principe résulter en une meilleure sensibilité de l'IF par rapport aux deux autres méthodes.¦Les anti-EBNA IgG peuvent disparaître chez les patients immunocompromis chez qui se produit une réactivation d'EBV. Nous avons donc recherché le status immunitaire des patients du groupe dont les sérums étaient négatifs pour anti-EBNA IgG en IF et positifs par les autres méthodes: seulement 28 des 70 patients étaient immunocompromis.¦Par conséquent, il est probable que dans la majorité de ces résultats discordants, les anticorps anti-EBNA IgG détectés par BBA et EIA sont de vrais positifs non décelés par l'IF.¦En conclusion, BBA est meilleur que la méthode de référance qu'est l'IF, et est égal à EIA en ce qui concerne la performance diagnostique. En outre, ces deux nouvelles méthodes offrent une économie de temps en raison de manipulations moindres, et ne requièrent aucune formation en microscopie à fluorescence. Elles sont également plus économes en échantillons que IF. BBA a l'avantage de n'avoir besoin que de deux analyses pour donner un diagnostique, alors que IF et EIA ont en besoin d'une par anticorps. Enfin, BBA dispose de contrôles internes permettant de reconnaître les liaisons non antigène-spécifiques des anticorps. Par contre, BBA nécessite l'achat d'un lecteur par cytométrie de flux assez coûteux.

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

Factors Supporting Cysteine Tolerance and Sulfite Production in Candida albicans.

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.

The Peroxisomal Enzyme L-PBE Is Required to Prevent the Dietary Toxicity of Medium-Chain Fatty Acids.

Specific metabolic pathways are activated by different nutrients to adapt the organism to available resources. Although essential, these mechanisms are incompletely defined. Here, we report that medium-chain fatty acids contained in coconut oil, a major source of dietary fat, induce the liver ω-oxidation genes Cyp4a10 and Cyp4a14 to increase the production of dicarboxylic fatty acids. Furthermore, these activate all ω- and β-oxidation pathways through peroxisome proliferator activated receptor (PPAR) α and PPARγ, an activation loop normally kept under control by dicarboxylic fatty acid degradation by the peroxisomal enzyme L-PBE. Indeed, L-pbe(-/-) mice fed coconut oil overaccumulate dicarboxylic fatty acids, which activate all fatty acid oxidation pathways and lead to liver inflammation, fibrosis, and death. Thus, the correct homeostasis of dicarboxylic fatty acids is a means to regulate the efficient utilization of ingested medium-chain fatty acids, and its deregulation exemplifies the intricate relationship between impaired metabolism and inflammation.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples

A total of 187 isolates from several clinical specimens were identified to species level as 129 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CNS) strains by the API Staph System (Biomerieux). Slime production was detected both by the conventional Christensen's method as well as by the Congo red agar method. Seventy-two strains of staphylococci isolates (38.5%) were found to be slime producers by Christensen's test tube method whereas 58 strains (31%) were slime positive with Congo red agar method. There was no statistically significant difference between the two methods for the detection of slime production (P > 0.05). Susceptibility of isolates against antimicrobial agents was tested by the disk diffusion method. Staphylococcal species had resistance to one or more antibiotics. Among the various antimicrobial agents, oxacillin (71.1%) and erythromycin (47.1%) showed higher resistance than most of the agents used against all isolates. Oxacillin resistant S. aureus (ORSA) and oxacillin resistant coagulase-negative staphylococci (ORCNS), 97 (75.2%) and 36 (62.1%) respectively were frequently observed in strains isolated from clinical materials. Among the ORSA strains, two strains were resistant to vancomycin. Moreover, 96 (74.4%) of 129 S. aureus strains were positive for blactamase enzyme. However, 78 (81.25%) of 96 b-lactamase positive S. aureus strains were b-lactamase positive ORSA isolates, but none of them had vancomycin resistance.

Connexin40 regulates renin production and blood pressure.

Renin secretion is regulated by coordinated signaling between the various cells of the juxtaglomerular apparatus. The renin-secreting cells (RSC), which play a major role in the control of blood pressure, are coupled to each other and to endothelial cells by Connexin40 (Cx40)-containing channels. In this study, we show that Cx40 knockout (Cx40-/-) mice, but not their heterozygous littermates, are hypertensive due to the increase in the number of RSC, renin biosynthesis, and plasma renin. Treatment with the angiotensin II receptor AT1 antagonist candesartan or the angiotensin II-converting enzyme inhibitor ramipril reduced the blood pressure of the Cx40-/- mice to the same levels seen in wild-type (WT) mice. The elevated blood pressure of the knockout mice was not affected by clipping one renal artery (2K1C, renin-dependent model of hypertension) or after a high salt diet. Under these conditions, however, Cx40-/- mice showed an altered production and release of renin. The renin mRNA ratio between the clipped and the non-clipped kidney was lower in the knockout than in the WT 2K1C mice. This indicates that the response to a change in blood pressure was altered. The RSC of the Cx40-/- mice did not have a compensatory increase in the levels of either Cx43 or Cx37. Our data show that renin secretion is dependent on Cx40 and suggest the Cx40-/- mice may be a genetic model of renin-dependent hypertension.

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Role of bradykinin in the neonatal renal effects of angiotensin converting enzyme inhibition.

The vascular effects of angiotensin converting enzyme inhibitors are mediated by the inhibition of the dual action of angiotensin converting enzyme (ACE): production of angiotensin II and degradation of bradykinin. The deleterious effect of converting enzyme inhibitors (CEI) on neonatal renal function have been ascribed to the elevated activity of the renin-angiotensin system. In order to clarify the role of bradykinin in the CEI-induced renal dysfunction of the newborn, the effect of perindoprilat was investigated in anesthetized newborn rabbits with intact or inhibited bradykinin B2 receptors. Inulin and PAH clearances were used as indices of GFR and renal plasma flow, respectively. Perindoprilat (20 microg/kg i.v.) caused marked systemic and renal vasodilation, reflected by a fall in blood pressure and renal vascular resistance. GFR decreased, while urine flow rate did not change. Prior inhibition of the B2 receptors by Hoe 140 (300 microg/kg s.c.) did not prevent any of the hemodynamic changes caused by perindoprilat, indicating that bradykinin accumulation does not contribute to the CEI-induced neonatal renal effects. A control group receiving only Hoe 140 revealed that BK maintains postglomerular vasodilation via B2 receptors in basal conditions. Thus, the absence of functional B2 receptors in the newborn was not responsible for the failure of Hoe 140 to prevent the perindoprilat-induced changes. Species- and/or age-related differences in the kinin-metabolism could explain these results, suggesting that in the newborn rabbit other kininases than ACE are mainly responsible for the degradation of bradykinin.