Improvement of a photoautotrophic chassis robustness for synthetic biology applications

Cyanobacteria are photoautotrophic microorganisms with great potential for the biotechnological industry due to their low nutrient requirements, photosynthetic capacities and metabolic plasticity. In biotechnology, the energy sector is one of the main targets for their utilization, especially to produce the so called third generation biofuels, which are regarded as one of the best replacements for petroleum-based fuels. Although, several issues could be solved, others arise from the use of cyanobacteria, namely the need for high amounts of freshwater and contamination/predation by other microorganisms that affect cultivation efficiencies. The cultivation of cyanobacteria in seawater could solve this issue, since it has a very stable and rich chemical composition. Among cyanobacteria, the model microorganism Synechocystis sp. PCC 6803 is one of the most studied with its genome fully sequenced and genomic, transcriptomic and proteomic data available to better predict its phenotypic behaviors/characteristics. Despite suitable for genetic engineering and implementation as a microbial cell factory, Synechocystis’ growth rate is negatively affected by increasing salinity levels. Therefore, it is important to improve. To achieve this, several strategies involving the constitutive overexpression of the native genes encoding the proteins involved in the production of the compatible solute glucosylglycerol were implemented, following synthetic biology principles. A preliminary transcription analysis of selected mutants revealed that the assembled synthetic devices are functional at the transcriptional level. However, under different salinities, the mutants did not show improved robustness to salinity in terms of growth, compared with the wild-type. Nevertheless, some mutants carrying synthetic devices appear to have a better physiological response under seawater’s NaCl concentration than in 0% (w/v) NaCl.

Acellular Gellan-gum based bilayered structures for the regeneration of osteochondral defects: a preclinical study

 In orthopaedics, the management and treatment of osteochondral (OC) defects remains an ongoing clinical challenge. Autologous osteochondral mosaicplasty has been used as a valid option for OC treatments although donor site morbidity remains a source of concern [1]. Engineering a whole structure capable of mimicking different tissues (cartilage and subchondral bone) in an integrated manner could be a possible approach to regenerate OC defects. In our group we have been proposing the use of bilayered structures to regenerate osteochondral defects [2,3]. The present study aims to investigate the pre-clinical performance of bilayered hydrogels and spongy-like hydrogels in in vivo  models (mice and rabbit, respectively), in both subcutaneous and orthotopic models. The bilayered structures were produced from Low Acyl Gellan Gum (LAGG) from Sigma-Aldrich, USA. Cartilage-like layers were obtained from a 2wt% LAGG solution. The bone-like layers were made of 2wt% LAGG with incorporation of hydroxyapatite at 20% and 30% (w/v). Hydrogels and spongy-like were subcutaneouly implanted in mice to evaluate the inflammatory response. Then, OC defects were induced in rabbit knee to create a critical size defect (4 mm diameter and 5 mm depth), and then hydrogels and sponges implanted. Both structures followed different processing methods. The hydrogels were injected allowing in situ  crosslinking. Unlike, the spongy-like were pre-formed by freeze-drying. The studies concerning subcutaneous implantation and critical size OC defect were performed for 2 and 4 weeks time, respectively. Cellular behavior and inflammatory responses were assessed by means of histology staining and biochemical function and matrix deposition by immunohistochemistry. Additionally, both OC structures stability and new cartilage and bone formation were evaluated by using vivo- computed tomography (Scanco 80). The results showed no acute inflammatory response for both approaches. New tissue formation and integration in the adjacent tissues were also observed, which present different characteristic behaviors when comparing hydrogels and sponges response. As future insights, a novel strategy for regeneration of OC defects can be designed encompassing both, hydrogels and spongy-like structures and cellular approaches. References: 1. Espregueira-Mendes J. et al. Osteochondral transplantation using autografts from the upper tibio-fibular joint for the treatment of knee cartilage lesions. Knee Surgery, Sports Traumatology, Arthroscopy 20,1136, 2012. 2. Oliveira JM. et al, Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells. Biomaterials 27, 6123, 2006. 3. Pereira D R. et al. Gellan Gum-Based Hydrogel Bilayered Scaffolds for Osteochondral Tissue Engineering. Key Engineering Materials 587, 255, 2013.

Fucoidan-based particles for diabetes mellitus treatment

Marine organisms are rich in a variety of materials with potential use in Tissue Engineering and Regenerative Medicine. One important example is fucoidan, a sulfated polysaccharide extracted from the cell wall of brown seaweeds.  Fucoidan is composed by L-fucose, sulfate groups and glucuronic acid. It has important bioactive properties such as anti-oxidative, anticoagulant, anticancer and reducing the blood glucose (1). In this work, the biomedical potential of fucoidan-based materials as drug delivery system was assessed by processing modified fucoidan (MFu) into particles by photocrosslinking using superamphiphobic surfaces and visible light. Fucoidan was modified by methacrylation reaction using different concentrations of methacrylate anhydride, namely 8% v/v (MFu1) and 12% v/v (MFu2). Further, MFu particles with and without insulin (5% w/v) were produced by pipetting a solution of 5% MFu with triethanolamine and eosin-y onto a superamphiphobic surface and then photocrosslinking using visible light (2). The developed particles were characterized to assess their chemistry, morphology, swelling behavior, drug release, insulin content and encapsulation efficiency. Moreover, the viability assays of fibroblast L929 cells in contact with MFu particles showed good adhesion and proliferation up to 14 days. Furthermore, the therapeutic potential of these particles using human beta cells is currently under investigation. Results obtained so far suggest that modified fucoidan particles could be a good candidate for diabetes mellitus therapeutic approaches.  

Postharvest shelf life extension of blueberries using a chitosan-based edible coating containing aloe vera juice

Poster

Effect of chitosan-Aloe vera coating on postharvest quality of blueberry (Vaccinium corymbosum) fruit

The present study was carried out to evaluate the effect of chitosan-based edible coatings with Aloe vera extract on the postharvest blueberry fruit quality during storage at 5 °C. Firstly, A. vera fractions (pulp and liquid) were extracted from leaves and evaluated in terms of antifungal and antioxidant capacities. The choice of the most adequate chitosan and A. vera fraction concentrations to be incorporated in coating formulation was made based on the wettability of the corresponding coating solutions. Coatings with 0.5% (w/v) chitosan + 0.5% (w/v) glycerol + 0.1% (w/v) Tween 80 + 0.5% (v/v) A. vera liquid fraction presented the best characteristics to uniformly coat blueberry surface. Physico-chemical (i.e., titratable acidity, pH, weight loss) and microbiological analyses of coated blueberries (non-inoculated or artificially inoculated with Botrytis cinerea) were performed during 25 d. Microbiological growth and water loss levels were approximately reduced by 50% and 42%, respectively, in coated blueberries after 25 d compared to uncoated blueberries. After 15 d, weight loss values were 6.2% and 3.7% for uncoated and chitosanA. vera coated blueberries, respectively. Uncoated fruits presented mold contamination after 2 d of storage (2.0 ± 0.32 log CFU g1), whilst fruits with chitosan-based coatings with A. vera presented mold contamination only after 9 d of storage (1.3 ± 0.35 log CFU g1). Overall, coatings developed in this study extend blueberries shelf-life for about 5 d, demonstrating for the first time that the combination of chitosan and A. vera liquid fraction as edible coating materials has great potential in expanding the shelf-life of fruits.

Gellan gum-hyaluronate spongy-like hydrogels promote angiogenesis in hindlimb ischemia

"Tissue engineering: part A", vol. 21, suppl. 1 (2015)

Chondrogenic potential of injectable κ-carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue-engineering applications

Due to the limited self-repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of k-carrageenan hydrogels for the delivery of stem cells obt ained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation met hod and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v k-carrageenan solution at a cell density of 5  10 6 cells/ml. The results from the analysis of the cell-encapsulating hydrogels, cultured for up to 21 days, indicated that k-carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mec hanical analysis demonstrated an increase in stiffness and viscoelastic properties of k-carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that k-carrageenan exhibits properties t hat enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

Valorization of agro-industrial wastes towards the production of rhamnolipids

In this work, oil mill wastewater (OMW), a residue generated during olive oil extraction, was evaluated as an inducer of rhamnolipid production. Using a medium containing as sole ingredients corn steep liquor (10%, v/v), sugarcane molasses (10%, w/v) and OMW (25%, v/v), Pseudomonas aeruginosa #112 produced 4.5 and 5.1 g of rhamnolipid per liter in flasks and reactor, respectively, with critical micelle concentrations as low as 13 mg/l. Furthermore, in the medium supplemented with OMW, a higher proportion of more hydrophobic rhamnolipid congeners was observed comparing with the same medium without OMW. OMW is a hazardous waste which disposal represents a serious environmental problem; therefore, its valorization as a substrate for the production of added-value compounds such as rhamnolipids is of great interest. This is the first report of rhamnolipid production using a mixture of these three agro-industrial by-products, which can be useful for the sustainable production of rhamnolipids.

Revue bryologique.

v.32-35 (1905-1908)

North American flora.

v. 22, pt. 3 (1908)

North American flora.

v. 22, pt. 4 (1908)

Report on the scientific results of the voyage of S.Y. "Scotia" during the years 1902, 1903 and 1904, under the leadership of William S. Bruce.

v. 3,pt.1-11

Contribuições à teoria da genética em populações

1) O equilíbrio em populações, inicialmente compostas de v��rios genotipos depende essencialmente de três fatores: a modalidade de reprodução e a relativa viabilidade e fertilidade dos genotipos, e as freqüências iniciais. 2) Temos que distinguir a) reprodução por cruzamento livre quando qualquer indiv��duo da população pode ser cruzado com qualquer outro; b) reprodução por autofecundação, quando cada indiv��duo é reproduzido por uma autofecundação; c) finalmente a reprodução mista, isto é, os casos intermediários onde os indiv��duos são em parte cruzados, em parte autofecundados. 3) Populações heterozigotas para um par de gens e sem seleção. Em populações com reprodução cruzada se estabelece na primeira geração um equilíbrio entre os três genotipos, segundo a chamada regra de Hardy- Weinberg. Inicial : AA/u + Aa/v aa/u = 1 Equilibirio (u + v/2)² + u + v/2 ( w + v/2) + (w + v/2)² = p2 + 2 p o. q o. + q²o = 1 Em populações com autofecundação o equilíbrio será atingido quando estiverem presentes apenas os dois homozigotos, e uma fórmula é dada que permite calcular quantas gerações são necessárias para atingir aproximadamente este resultado. Finalmente, em populações com reprodução mista, obtemos um equilíbrio com valores intermediários, conforme Quadro 1. Frequência Genotipo Inicial mº Geração Final AA u u + 2m-1v / 2m+1 u + 1/2v Aa v 2/ 2m+2 v - aa w w + 2m - 1/ 2m + 1 v w + 1/2 v 4) Os índices de sobrevivencia. Para poder chegar a fórmulas matemáticas simples, é necessário introduzir índices de sobreviv��ncia para medir a viabilidade e fertilidade dos homozigotos, em relação à sobreviv��ncia dos heterozigotos. Designamos a sobreviv��ncia absoluta de cada um dos três genotipos com x, y e z, e teremos então: x [ A A] : y [ Aa] : z [ aa] = x/y [ A A] : [ Aa] : z/ y [aa] = R A [ AA] : 1 [Aa] : Ra [aa] É evidente que os índices R poderão ter qualquer valor desde zero, quando haverá uma eliminação completa dos homozigotos, até infinito quando os heterozigotos serão completamente eliminados. Os termos (1 -K) de Haldane e (1 -S) ou W de Wright não têm esta propriedade matemática, podendo variar apenas entre zero e um. É ainda necessário distinguir índices parciais, de acordo com a marcha da eliminação nas diferentes fases da ontogenia dos indiv��duos. Teremos que distinguir em primeiro lugar entre a eliminação durante a fase vegetativa e a eliminação na fase reprodutiva. Estas duas componentes são ligadas pela relação matemática. R - RV . RR 5) Populações com reprodução cruzada e eliminação. - Considerações gerais. a) O equilibrio final, independente da freqüência inicial dos genes e dos genotipos para valores da sobreviv��ncia diferentes de um, é atingido quando os gens e os genotipos estão presentes nas proporções seguintes: (Quadro 2). po / qo = 1- ro / 1-Ra [AA] (1 - Ro)² . Rav [ Aa] = 2(1 - Ra) ( 1 - Ra) [a a} = ( 1 - Ra)² . RaA b) Fórmulas foram dadas que permitem calcular as freqüências dos genotipos em qualquer geração das populações. Não foi tentado obter fórmulas gerais, por processos de integração, pois trata-se de um processo descontínuo, com saltos de uma e outra geração, e de duração curta. 6) Populações com reprodução cruzada e eliminação. Podemos distinguir os seguintes casos: a) Heterosis - (Quadro 3 e Fig. 1). Ra < 1; Ra < 1 Inicial : Final : p (A)/q(a) -> 1-ra/1-ra = positivo/zero = infinito Os dois gens e assim os três genotipos zigóticos permanecem na população. Quando as freqüências iniciais forem maiores do que as do equilíbrio elas serão diminuidas, e quando forem menores, serão aumentadas. b) Gens recessivos letais ou semiletais. (Quadro 1 e Fig. 2). O equilíbrio será atingido quando o gen, que causa a redução da viabilidade dos homozigotos, fôr eliminado da população. . / c) Gens parcialmente dominantes semiletais. (Quadro 5 e Fig. 3). Rª ; Oz Ra < 1 Inicial : Equilibrio biológico Equilíbrio Matemático pa(A)/q(a) -> positivo /zero -> 1- Rq/ 1-Ra = positivo/negativo d) Genes incompatíveis. Ra > 1 ; Ra > 1; Ra > Ra Equílibrio/biológico p (A)/ q(a) -> positivo/zero Equilibrio matemático -> positivo/ zero -> zero/negativo -> 1-Ra/1 - Ra = negativo/negativo Nestes dois casos devemos distinguir entre o significado matemático e biológico. A marcha da eliminação não pode chegar até o equilíbrio matemático quando um dos gens alcança antes a freqüência zero, isto é, desaparece. Nos três casos teremos sempre uma eliminação relativamente rápida de um dos gens «e com isso do homozigoto respectivo e dos heterozigotòs. e) Foram discutidos mais dois casos especiais: eliminação reprodutiva diferencial dos dois valores do sexo feminino e masculino, -e gens para competição gametofítica. (Quadros 6 e 7 e Figs. 4 a 6). 7) População com autofecundação e seleção. O equilíbrio será atingido quando os genotipos estiverem presentes nas seguintes proporções: (Quadro 8); [AA] ( 0,5 - Ra). R AV [Aa] = 4. ( 0,5 - Ra) . (0.5 -R A) [aa] ( 0,5 - R A) . Rav Também foram dadas fórmulas que permitem calcular as proporções genotípicas em cada geração e a marcha geral da eliminação dos genotipos. 8)Casos especiais. Podemos notar que o termo (0,5 -R) nas fórmulas para as populações autofecundadas ocupa mais ou menos a mesma importância do que o termo (1-R) nas fórmulas para as populações cruzadas. a) Heterosis. (Quadro 9 e Fig. 7). Quando RA e Ra têm valores entre 0 e 0,5, obtemos o seguinte resultado: No equilíbrio ambos os gens estão presentes e os três heterozigotos são mais freqüentes do que os homozigotos. b) Em todos os demais casos, quando RA e Ra forem iguais ou maiores do que 0,5, o equilíbrio é atingido quando estão representados na população apenas os homozigotos mais viáveis e férteis. (Quadro 10). 9) Foram discutidos os efeitos de alterações dos valores da sobreviv��ncia (Fig. 9), do modo de reprodução (Fig. 10) e das freqüências iniciais dos gens (Fig. 8). 10) Algumas aplicações à genética aplicada. Depois de uma discussão mais geral, dois problemas principais foram tratados: a) A homogeneização: Ficou demonstrado que a reprodução por cruzamento livre representa um mecanismo muito ineficiente, e que se deve empregar sempre ou a autofecundação ou pelo menos uma reprodução mista com a maior freqüência possível de acasalamentos consanguíneos. Fórmulas e dados (Quadro 11 e 12), permitem a determinação do número de gerações necessárias para obter um grau razoável de homozigotia- b) Heterosis. Existem dois processos, para a obtenção de um alto grau de heterozigotia e com isso de heterosis: a) O método clássico do "inbreeding and outbreeding". b) O método novo das populações balançadas, baseado na combinação de gens que quando homozigotos dão urna menor sobreviv��ncia do que quando heterozigotos. 11) Algumas considerações sobre a teoria de evolução: a) Heterosis. Os gens com efeito "heterótico", isto é, nos casos onde os heterozigotos s mais viáveis e férteis, do que os homozigotos, oferecem um mecanismo especial de evolução, pois nestes casos a freqüência dos gens, apesar de seu efeito negativo na fase homozigota, tem a sua freqüência aumentada até que seja atingido o valor do equilíbrio. b) Gens letais e semiletais recessivos. Foi demonstrado que estes gens devem ser eliminados automáticamente das populações. Porém, ao contrário do esperado, não s raros por exemplo em milho e em Drosophila, gens que até hoje foram classificados nesta categoria. Assim, um estudo detalhado torna-se necessário para resolver se os heterozigotos em muitos destes casos não serão de maior sobreviv��ncia do que ambos os homozigotos, isto é, que se trata realmente de genes heteróticos. c) Gens semiletais parcialmente dominantes. Estes gens serão sempre eliminados nas populações, e de fato eles são encontrados apenas raramente. d) Gens incompatíveis. São também geralmente eliminados das populações. Apenas em casos especiais eles podem ter importância na evolução, representando um mecanismo de isolamento.