Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions

The development of versatile bioactive surfaces able to emulate in vivo conditions is of enormous importance to the future of cell and tissue therapy. Tuning cell behaviour on two-dimensional surfaces so that the cells perform as if they were in a natural three-dimensional tissue represents a significant challenge, but one that must be met if the early promise of cell and tissue therapy is to be fully realised. Due to the inherent complexities involved in the manufacture of biomimetic three-dimensional substrates, the scaling up of engineered tissue-based therapies may be simpler if based upon proven two-dimensional culture systems. In this work, we developed new coating materials composed of the self-assembling peptide amphiphiles (PAs) C16G3RGD (RGD) and C16G3RGDS (RGDS) shown to control cell adhesion and tissue architecture while avoiding the use of serum. When mixed with the C16ETTES diluent PA at 13 : 87 (mol mol-1) ratio at 1.25 times 10-3 M, the bioactive {PAs} were shown to support optimal adhesion, maximal proliferation, and prolonged viability of human corneal stromal fibroblasts ({hCSFs)}, while improving the cell phenotype. These {PAs} also provided stable adhesive coatings on highly-hydrophobic surfaces composed of striated polytetrafluoroethylene ({PTFE)}, significantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. The thickness and structure of this highly-organised tissue were similar to those observed in vivo, comprising aligned newly-deposited extracellular matrix. As such, the developed coatings can constitute a versatile biomaterial for applications in cell biology, tissue engineering, and regenerative medicine requiring serum-free conditions.

Effect of breadmaking process on In Vitro gut microbiota parameters in irritable bowel syndrome

A variety of foods have been implicated in symptoms of patients with Irritable Bowel Syndrome (IBS) but wheat products are most frequently cited by patients as a trigger. Our aim was to investigate the effects of breads, which were fermented for different lengths of time, on the colonic microbiota using in vitro batch culture experiments. A set of in vitro anaerobic culture systems were run over a period of 24 h using faeces from 3 different IBS donors (Rome Criteria–mainly constipated) and 3 healthy donors. Changes in gut microbiota during a time course were identified by fluorescence in situ hybridisation (FISH), whilst the small -molecular weight metabolomic profile was determined by NMR analysis. Gas production was separately investigated in non pH-controlled, 36 h batch culture experiments. Numbers of bifidobacteria were higher in healthy subjects compared to IBS donors. In addition, the healthy donors showed a significant increase in bifidobacteria (P<0.005) after 8 h of fermentation of a bread produced using a sourdough process (type C) compared to breads produced with commercial yeasted dough (type B) and no time fermentation (Chorleywood Breadmaking process) (type A). A significant decrease of δ-Proteobacteria and most Gemmatimonadetes species was observed after 24 h fermentation of type C bread in both IBS and healthy donors. In general, IBS donors showed higher rates of gas production compared to healthy donors. Rates of gas production for type A and conventional long fermentation (type B) breads were almost identical in IBS and healthy donors. Sourdough bread produced significantly lower cumulative gas after 15 h fermentation as compared to type A and B breads in IBS donors but not in the healthy controls. In conclusion, breads fermented by the traditional long fermentation and sourdough are less likely to lead to IBS symptoms compared to bread made using the Chorleywood Breadmaking Process.

Improved method for ex ovo-cultivation of developing chicken embryos for human stem cell xenografts

The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26-HH28/E5-E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.

An in vitro study of the effect of probiotics, prebiotics and synbiotics on the elderly faecal microbiota

The use of dietary intervention in the elderly in order to beneficially modulate their gut microbiota has not been extensively studied. The influence of two probiotics (Bifidobacterium longum and Lactobacillus fermentum) and two prebiotics [isomaltooligosaccharides (IMO) and short-chain fructooligosaccharides (FOS)], individually and in synbiotic combinations (B. longum with IMO, L. fermentum with FOS) on the gut microbiota of elderly individuals was investigated using faecal batch cultures and three-stage continuous culture systems. Population changes of major bacterial groups were enumerated using fluorescent in situ hybridisation (FISH). B. longum and IMO alone significantly increased the Bifidobacterium count after 5 and 10 h of fermentation and their synbiotic combination significantly decreased the Bacteroides count after 5 h of fermentation. L. fermentum and FOS alone significantly increased the Bifidobacterium count after 10 h and 5, 10 and 24 h of fermentation respectively. B. longum with IMO as well as B. longum and IMO alone significantly increased acetic acid concentration during the fermentation in batch cultures. In the three-stage continuous culture systems, both synbiotic combinations increased the Bifidobacterium and Lactobacillus count in the third vessel representing the distal colon. In addition, the synbiotic combination of L. fermentum with scFOS resulted in a significant increase in the concentration of acetic acid. The results show that the elderly gut microbiota can be modulated in vitro with the appropriate pro-, pre- and synbiotics.

Estudo comparativo de dois tipos de cultivo: monocultivo (camarões) versus cultivo integrado (algas/camarões)

The integrated culture of seaweed and aquatic animals is an ancient practice in Asian countries. The expansion of this practice to western countries is consequence of the recognition of this system as a sustainable alternative that allows economical diversification and mitigation of environmental impacts generated by effluents of aquaculture. This study evaluated the growth of the seaweed Gracilaria caudata and of the shrimp Litopenaeus vannamei in monoculture (shrimps) and integrated culture (shrimps and algae) systems, and accessed the effect of the seaweed in the water quality. There were two treatments in the experiment: monoculture (shrimps) and integrated culture (shrimps/ algae). The organisms were cultured in 6 aquaria (10L) filled with seawater (35.0±0.0 PSU and 28.1±0.4°C) for 28 days. The nutrients of water (PO43-, NH4+, NO2-, NO3- and DIN), the biomass and the relative growth rate (RGR, % day-1) of seaweed and shrimps were measured weekly. The parameters pH, temperature, salinity and dissolved oxygen were measured daily. The concentration of NH4+ in integrated culture (62.8±25.2µM) was lower (Mann-Whitney p<0.001) than in monoculture (85.6±24.3µM). The mean of PO4- in monoculture (10.4±4.6µM) was markedly higher (Mann-Whitney; p=0.024) than that in integrated culture (8.7±4.1µM). The level of dissolved oxygen in integrated culture (6.0±0.6mg/L) was higher (t-Student; P=0.014) than that in shrimp monoculture (5.8±0.6mg/L). The mean values of the parameters pH, NO2-, NO3- and DIN were 7.5±0.2, 10.1±12.2µM, 24.5±3.2µM and 120.17±30.76µM in monoculture, and 7.5±0.2, 10.5±13.2µM, 27.4±3.5µM and 100.76±49.59µM in integrated culture. There were not differences in these parameters between treatments. The biomass and RGR of seaweed reached 15.0±1.9g and 7.4±2.8% day-1 at the end of the experiment. The performance of shrimp was favorable in monoculture (1.5±0.8g; 5.7±1.6% dia-1) and in integrated culture (1.5±0.7g; 5.2±1.2% dia-1), and the rate of survival was 100% in both treatments. The tolerance and favorable performance of Gracilaria caudata suggest that this seaweed might be integrated into shrimp (Litopenaeus vannamei) culture systems

Influence of stocking densities in the nursery phase on the growth of Macrobrachium amazonicum reared in net pens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Desempenho produtivo de Pseudoplatystoma corruscans estocados em sistemas de criação: semi-intensivo (viveiro escavado) e intensivo (tanque-rede)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Population growth and development of two species of Cladocera, Moina micrura and Diaphanosoma birgei, in laboratory

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Steroidogenesis in cumulus cells of bovine cumulus-oocyte-complexes matured in vitro with BSA and different concentrations of steroids

The present in vitro experiments were designed to evaluate the ability of bovine cumulus-oocyte-complexes (COCs)to produce steroids and also to evaluate the modulatory effects of added estradiol, progesterone and testosterone on the steroidogenic activity of COCs. Considerable estradiol accumulation was observed in the control maturation medium for in vitro maturation of bovine COCs during the 24h of maturation (P < 0.05). When testosterone was added to the medium at various concentrations, a slight estradiol accumulation occurred, which, however, was lower (P < 0.05) than that observed in the control medium. Slight estradiol accumulation was observed in maturation medium containing progesterone at concentrations of 2.5, 5.0 and 10.0 mug/ml, but these increases were less (P < 0.05) than those observed in the control medium. However, in the presence of 1.0 mug/ml progesterone, estradiol accumulation was equal to that of the control medium (P > 0.05). Progesterone accumulation (P < 0.05) was observed in the control medium for in vitro maturation of bovine COCs. When estradiol was added to the maturation medium, progesterone accumulation was observed, but was significant (P < 0.05) only when the medium was supplemented with the lesser concentrations of estradiol utilized in the experiment (1.0 mug/ml). The results demonstrated that (1) cumulus cells of bovine COCs are able to secrete estradiol and progesterone in culture systems for in vitro maturation, and this steroidogenesis is modulated by the steroids progesterone, testosterone and estradiol, and (2) the addition of estradiol to the in vitro maturation medium of bovine oocytes should be reviewed, since cumulus cells of COCs have been demonstrated to secrete estradiol in the maturation medium. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Sistemas de cultivo e uso de diferentes adubos na produção de silagem e grãos de milho

O presente trabalho teve por objetivo avaliar o desempenho do milho para produção de silagem e grãos, cultivado em diferentes sistemas e adubações. O delineamento experimental adotado foi de blocos inteiramente casualizados, em esquema fatorial 2 x 3, composto por dois sistemas de cultivo (sucessão e rotação de culturas) e três fontes de adubação (orgânica, mineral e organomineral), com quatro repetições. Para produção de silagem de planta inteira, os sistemas e as adubações não diferiram estatisticamente entre si (p < 0,05). Para produção de silagem de grão úmido, o sistema em sucessão e a adubação mineral mostraram-se superiores aos demais, produzindo 10.823 e 10.815 kg ha-1, respectivamente, não incrementando, contudo, qualidade à silagem. Já na colheita dos grãos, o sistema em sucessão produziu em média 6.820 kg ha-1 e foi superior ao sistema em rotação. A adubação mineral foi superior às demais, produzindo em média 7.277 kg ha-1, contudo apresentou 54,62% de plantas acamadas, fato este que resultaria em perdas significativas na produtividade, caso a colheita tivesse sido mecanizada.

Glomus clarum e G. etunicatum: cultivo em solo e aeroponia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Nutrição e produtividade da cultura do milho em sistemas de culturas e fontes de adubação

A utilização de esterco animal na agricultura, aliada ao cultivo de plantas de cobertura, pode conferir sustentabilidade ao sistema agrícola. Com o objetivo de avaliar o efeito de dois sistemas de culturas e de três fontes de nutrientes, na cultura do milho, conduziu-se um experimento, em Latossolo Vermelho, no município de Marechal Cândido Rondon (PR). O delineamento experimental utilizado foi de blocos casualizados, com quatro repetições, e, os tratamentos, dispostos em esquema fatorial 2 x 3, compostos por dois sistemas de culturas (sucessão trigo/milho e consórcio aveia+ervilhaca+nabo/milho) e três fontes de nutrientes (mineral, orgânica e organomineral). em junho de 2006, implantou-se a cultura do trigo e o consórcio de plantas de cobertura. em outubro de 2006, semeou-se milho sob os dois sistemas. As adubações orgânica e organomineral consistiram na aplicação de dejetos de suínos, unicamente, e combinada com fertilizante mineral, respectivamente. O consórcio de aveia preta, ervilhaca peluda e nabo forrageiro mostrou-se capaz de fornecer a quantidade adequada de biomassa, concretizando sua viabilidade para produção de cobertura vegetal ao solo, durante o inverno. O cultivo de milho em sucessão ao trigo proporcionou a maior absorção de N e K e a maior produtividade. A adubação mineral proporcionou maior absorção de N e maior produtividade de milho, em comparação com as adubações orgânica e organomineral com dejetos de suínos.

Efeitos de diferentes sistemas de cultivo in vitro sobre o crescimento de folículos pré-antrais isolados de ovários de fetos bovinos

The objective of this study is to use different in vitro culture systems of preantral follicles from Nelore breed bovine fetuses in the last gestation quarter. The evaluation of treatments considered the time of growth of isolated follicles. Preantral follicles were mechanically isolated and submitted to the individual culture, for 9 days, in media no supplemented or supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or synthetic defined supplement substitute of serum KnockoutSR (KNO). We have also evaluated the effects of collagen gel or fetal calf fibroblast monolayer as substratum for in vitro cultures. The increase on the follicular diameter was followed in the first day (0 h), at the 72 h, 144 h and 216 h. Considering cultures of isolated follicles, the results have shown that the association between media supplemented with FCS and collagen gel was significantly more efficient on the increase of the follicular diameter than other treatments. It is not still established a system of appropriate cultivation that sustains the differentiation and multiplication of the granular cells and that maintains the contact of the same ones with the oocyte to provide molecules and factors that supply the metabolic demand. We also understand that our results also represent another promising step on the search for the ultimate system of in vitro culture of preantral follicles from bovines.

Reproductive technology in domestic carnivorous

Background: The delay in development of artificial reproduction techniques on carnivorous could be due to countless reasons, but the lack of commercial interest is probably the most important one. The majority of canines are small structures, canidae are extremely fertile and a great number of species are adapted to domestication or captivity. Finally, the canine gamete physiology presents a difficult adaptation of technology knowledge obtained from other species. Furthermore, domestic felines are animals of company and there is no interest in reproducing them in a large scale, as it has been observed in other domestic animals, however, besides of being a valuable model for the development of in vitro techniques, the domestic cat is also used as an embryo receptor for different species of small wild felines due to physiological similarities among them, in vitro embrionary development, Review: It was reviewed the main insights about the reproductive physiology in female dogs, in vitro oocytary maturation (IVM), pregnancy and conception rate with dogs' frozen/unfrozen semen and PIV in domestic cats. The majority of mammal oocytes restart meiosis spontaneously after ovulation and reaches MII in artificial environment; in an in vitro maturation system in bovines, around 90% of oocytes complete their maturation, although its development capacity can be reduced subsequently. The success of IVM in canidae have been limited, with maturation rate varying from 0 to 58%, usually around 20%. The greatest difficulties include oocyte quality, hormonal environment, protein supplementation, cumulus / oocyte cell interaction, donor breed and age, culture systems, oxygen tension, amino acids, growth factor and sequential means. The freezing process reduces the quality of the semen, firstly because it reduces the number of living sperms and secondly because freezing produces cell modifications that could alter the sperm motility, longevity, integrity of membranes and its fertilizing capacity. Conclusion: Nowadays, several researches are being performed with the aim of increasing viability after dogs' and cats' semen is unfrozen, using extenders, cryoprotectors, freezing and unfreezing curves, addition of antioxidant substances. The aim of this text is to inform about the improvements obtained on the artificial reproduction techniques, emphasizing the oocytary maturation in female dogs, semen cryopreservation and artificial insemination in domestic dogs and cats.

Ontogenia do sistema sensorial de pacu Piaractus mesopotamicus (Holmberg, 1887) (Characidae: Serrasalmidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)