Amyloid-beta oligomers regulate the properties of human neural stem cells through GSK-3beta signaling

Alzheimer's disease (AD) is the most common cause of age-related dementia. The neuropathological hallmarks of AD include extracellular deposition of amyloid-beta peptides and neurofibrillary tangles that lead to intracellular hyperphosphorylated tau in the brain. Soluble amyloid-beta oligomers are the primary pathogenic factor leading to cognitive impairment in AD. Neural stem cells (NSCs) are able to self-renew and give rise to multiple neural cell lineages in both developing and adult central nervous systems. To explore the relationship between AD-related pathology and the behaviors of NSCs that enable neuroregeneration, a number of studies have used animal and in vitro models to investigate the role of amyloid-beta on NSCs derived from various brain regions at different developmental stages. However, the Abeta effects on NSCs remain poorly understood because of conflicting results. To investigate the effects of amyloid-beta oligomers on human NSCs, we established amyloid precursor protein Swedish mutant-expressing cells and identified cell-derived amyloid-beta oligomers in the culture media. Human NSCs were isolated from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres. Human NSCs exposure to cell-derived amyloid-beta oligomers decreased dividing potential resulting from senescence through telomere attrition, impaired neurogenesis and promoted gliogenesis, and attenuated mobility. These amyloid-beta oligomers modulated the proliferation, differentiation and migration patterns of human NSCs via a glycogen synthase kinase-3beta-mediated signaling pathway. These findings contribute to the development of human NSC-based therapy for AD by elucidating the effects of Abeta oligomers on human NSCs.

Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol

The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Optimización de la producción de biomasa usando glicerol crudo, de una cepa mutante de Yarrowia lipolytica con actividad incrementada de lipasa. La levadura Yarrowia lipolytica acumula aceites y produce una lipasa extracelular al crecer en diferentes fuentes de carbono, entre ellas el glicerol, principal subproducto de la creciente industria del biodiésel. En el presente trabajo, se optimizó mediante la metodología de superficies de respuesta la producción de biomasa de una nueva cepa mutante de Y. lipolytica, empleando medios con glicerol derivado de la industria del biodiésel como principal fuente de carbono. Esta cepa presentó características morfológicas y perfil de ácidos grasos distintivos, y una mayor actividad de lipasa extracelular. Para obtener una producción significativa de lipasa extracelular, fue necesario el agregado de una fuente orgánica de nitrógeno y de 1 g/l de aceite de oliva. Se utilizaron los diseños estadísticos de Plackett-Burman y central compuesto para la selección y la optimización de las fermentaciones en frascos agitados; los máximos valores de biomasa y de lipasa obtenidos fueron de 16,1 g/l y 12,2 unidades/ml, respectivamente. Luego, el bioproceso en lote optimizado se escaló a biorreactores aireados, y los valores de actividad específica de lipasa alcanzados después de haberse consumido el 95 % del glicerol fueron tres veces más altos que los obtenidos en los cultivos en frascos agitados. En suma, se desarrolló un bioproceso sostenible para la obtención de biomasa y de una actividad de lipasa extracelular, que a la vez maximiza el uso de subproductos de la industria del biodiésel.

Cultivo contínuo de Arthrospira (Spirulina) platensis em fotobiorreator tubular utilizando uréia como fonte de nitrogênio e CO2 puro ou proveniente de fermentação alcoólica / Continuous cultivation of Arthrospira (Spirulina) platensis in tubular photobioreactor using urea as nitrogen source and CO2 from cylinder or produced by alcoholic fermentation

A aplicabilidade do processo de produção de microrganismos fotossintetizantes depende da obtenção de altas concentrações de biomassa e para isso seria interessante o emprego de fotobiorreatores tubulares. Eles permitem redução da área de cultivo e menor perda de CO2 e nitrogênio amoniacal por volatilização. Em uma primeira etapa deste trabalho, Arthrospira platensis foi cultivada por processo contínuo, avaliando-se diferentes valores de vazão específica de alimentação (D = 0,2 a 1,0 dia-1) e diferentes intensidades luminosas (I = 60 e 120 µmol fótons.m-2.s-1). Verificou-se que 120 µmol fótons.m-2.s-1 associada a D igual a 0,2 dia-1 resultou em maior valor de concentração celular em regime permanente (XP = 2446 ± 74 mg.L-1.d-1), mas o mesmo I associado a maior valor de D (0,6 dia-1) levou ao melhor valor de produtividade em células (PX = 938,73 mg.L-1.d-1). Foi possível a obtenção do regime permanente em quase todos os ensaios, o que indica que o cultivo contínuo de A. platensis em fotobiorreator tubular, usando uréia como fonte de nitrogênio, pode levar a resultados satisfatórios. Considerando a preocupação em relação à substituição de combustíveis fósseis por biocombustíveis, é iminente o crescente aumento da produção de etanol ainda nos próximos anos, e esse trabalho propõe o uso do CO2 liberado pela fermentação alcoólica na produção de microrganismos fotossintetizantes como A. platensis. Para isso, em uma segunda etapa, A. platensis foi cultivada por processo contínuo, com I igual a 120 µmol fótons.m-2.s-1, empregando uréia e CO2 proveniente de fermentação alcoólica para manutenção de pH e reposição da fonte de carbono. O uso desse CO2, sem tratamento prévio, associado a D igual a 0,6 dia-1 e concentração de uréia de 3,2 mM no meio de alimentação, permitiu a obtenção de PX igual a 839 ± 25 mg.L-1.d-1, o que está próximo de 938 ± 30mg.L-1.d-1, obtido com CO2 puro de cilindro. Estes resultados mostram que o uso de CO2 de fermentação alcoólica, associado a...

Appropriately designed tubular photobioreactors seem to be suitable for photosynthetic biomass production. It can reduce the cultivation area and provide lower loss of CO2 and ammoniacal nitrogen by volatilization. In a first step of this study, Arthrospira platensis was cultivated by continuous process, testing different values of dilution rate (D = 0.2 to 1.0 d-1) and light intensities (I = 60 and 120 µmol photons.m-2.s-1). The results of these runs showed that the maximum steady-state cell concentration (XS = 2446 ± 74 mg.L-1.d-1) was achieved at 120 µmol photons.m-2.s-1 and D of 0.2 d-1, but the same light intensity associated to higher dilution rate (0.6 d-1) provided the highest cell productivity (PX = 938 ± 30 mg.L-1.d-1), a value appreciably higher than that reported in other studies. Besides, steady-state conditions were achieved in most of the runs indicating that A. platensis continuous cultivation in the tubular photobioreactor, using urea as nitrogen source, can be performed effectively, thus appearing an interesting alternative for the large scale fixation of carbon dioxide to mitigate the green house effect. Taking into account the concern about the substitution of fossil fuel with biofuels, its evident that the ethanol production is going to increase even more in the next years, and this study propose the use of the CO2 released by the alcoholic fermentation for the production of photosynthetic microorganism such as A. platensis. For this purpose, in a second step, cultivations of A. platensis were carried out with 120 µmol photons.m-2.s-1 by continuous process, using urea and CO2 from Alcoholic fermentation for pH maintenance and carbon source replacement. The use of this CO2, without any treatment, associated with a D of 0.6 d-1 and feed urea concentration of 3.2 mM provide us a PX of 839 ± 25 mg.L-1.d-1, which is slightly lower than 938 ±30 mg.L-1.d-1, obtained with pure CO2 from cylinder. Our results showed that the use of CO2 from...

Regulation of expression of matrix metalloproteinase-9 by JNK in Raw 264.7 cells: presence of inhibitory factor(s) suppressing MMP-9 induction in serum and conditioned media

Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/chemokines. Whereas the MEK-ERK and PI3K-Akt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-alpha, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125-mediated MMP-9 induction, similar to IFN-gamma. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-gamma. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression.

Cytotoxicity of yellow sand in lung epithelial cells.

The present study was carried out to observe the cytotoxicity of yellow sand in comparison with silica and titanium dioxide in a rat alveolar type II cell line (RLE-6TN). Yellow sand (China Loess) was obtained from the loess layer in the Gunsu Province of China. The mean particle diameter of yellow sand was about 0.003 +/- 0.001 mm. Major elements of yellow sand were Si(27.7 +/- 0.6%), Al(6.01 +/- 0.17%), and Ca(5.83 +/- 0.23%) in that order. Silica and yellow sand significantly decreased cell viability and increased [Ca2+]i. All three particles increased the generation of H2O2. TiO2 did not change Fenton activity, while silica induced a slight increase of Fenton activity. In contrast, yellow sand induced a significant increase of Fenton activity. Silica, yellow sand and TiO2 induced significant nitrite formations in RLE-6TN cells. Silica showed the highest increase in nitrite formation, while yellow sand induced the least formation of nitrite. Silica and yellow sand increased the release of TNF-a. Based on these results, we suggest that yellow sand can induce cytotoxicity in RLE-6TN cells and reactive oxygen species, Fenton activity and reactive nitrogen species might be involved in this toxicity.

Isolation & characterization of angiogenic factors from cultured cells & conditioned media.

Angiogenic factors were isolated by ion exchange chromatography from three established cell lines viz., HEp 2, HeLa and CHO, a primary culture of mouse mammary adenocarcinoma and from conditioned media of HEp2 and cultured mammary adenocarcinoma cells. The angiogenic activity was assayed by chicken chorioallantoic membrane assay. The angiogenic factor eluted at 0.5 M, exhibited lambda max at 258 +/- 1 nm, contained protein and nucleic acid. The cellular angiogenic factor showed a ratio of 1:1 for protein and nucleic acid whereas the secreted angiogenic factor had 3-5 parts of protein to 1 part of nucleic acid. The angiogenic factor from HEp2 and CHO cells did not bind to heparin-agarose. Microheterogeneity of the angiogenic factors was established by SDS-PAGE. Antiserum raised against the cellular angiogenic factor from HEp2 cells, showed a titre of 1:1600 by ELISA. The angiogenic factor was directly localized on whole cells by ELISA. Cellular as well as secreted angiogenic factors crossreacted with the antibody. Neutralizing effect of the antiserum on induction of angiogenesis was detected on chicken chorioallantoic membrane.