Islet amyloid polypeptide (IAPP, amylin) is the major protein component of the islet amyloid deposits associated with type 2 diabetes. The polypeptide lacks a well-defined structure in its monomeric state but readily assembles to form amyloid. Amyloid fibrils formed from TAPP, intermediates generated in the assembly of IAPP amyloid, or both are toxic to beta-cells, suggesting that islet amyloid formation may contribute to the pathology of type 2 diabetes. There are relatively few reported inhibitors of amyloid formation by IAPP. Here we show that the tea-derived flavanol, (-)-epigallocatechin 3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate] (EGCG), is an effective inhibitor of in vitro IAPP amyloid formation and disaggregates preformed amyloid fibrils derived from IAPP. The compound is thus one of a very small set of molecules which have been shown to disaggregate TAPP amyloid fibrils. Fluorescence-detected thioflavin-T binding assays and transmission electron microscopy confirm that the compound inhibits unseeded amyloid fibril formation as well as disaggregates IAPP amyloid. Seeding studies show that the complex formed by IAPP and EGCG does not seed amyloid formation by LAPP. In this regard, the behavior of IAPP is similar to the reported interactions of A beta and alpha-synuclein with EGCG. Alamar blue assays and light microscopy indicate that the compound protects cultured rat INS-1 cells against IAPP-induced toxicity. Thus, EGCG offers an interesting lead structure for further development of inhibitors of IAPP amyloid formation and compounds that disaggregate IAPP amyloid.
Biodegradable nanofibers have become a popular candidate for tissue engineering scaffolds because of their biomimetic structure that physically resembles the extracellular matrix. For certain tissue regeneration applications, prolonged in vitro culture time for cellular reorganization and tissue remodeling may be required. Therefore, extensive understanding of cellular effects on scaffold degradation is needed. There are only few studies on the degradation of nanofibers, and also the studies on degradation throughout cell culture are rare. In this study, polyglycolide (PGA), poly(DL-lactide-co-glycolide) (PLGA) and poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] were electrospun into nanofibrous meshes. The nanofibers were cultured with porcine smooth muscle cells for up to 3 months to evaluate their degradation behavior and cellular response. The results showed that the degradation rates are in the order of PGA >> PLGA > P(LLA-CL). PGA nanofibers degraded in 3 weeks and supported cell growth only in the first few days. PLGA nanofiber scaffolds facilitated cell growth during the first 30 days after seeding, but cell growth was slow thereafter. P(LLA-CL) nanofibers facilitated long-term (1-3 months) cell growth. mRNA quantification using real-time polymerase chain reaction revealed that some smooth muscle cell markers (a-actinin and calponin) and extracellular matrix genes (collagen and integrin) seemed to be downregulated with increased cell culture time. Cell culture significantly increased the degradation rate of PGA nanofibers, whereas the effect on PLGA and P(LLA-CL) nanofibers was limited. We found that the molecular weight of P(LLA-CL) and PLGA nanofibers decreased linearly for up to 100 days. Half lives of PLGA and P(LLA-CL) nanofibers were shown to be 80 and 110 days, respectively. In summary, this is the first study to our knowledge to evaluate long-term polymeric nanofiber degradation in vitro with cell culture. Cell culture accelerated the nanofibrous scaffold degradation to a limited extent. P(LLA-CL) nanofibers could be a good choice as scaffolds for long-term smooth muscle cell culture.
Chemical composition, nutritional value and other physico-chemical parameters of sea bass from two different geographical areas (Greece and Spain) and from aquaculture and wild origin were studied. Farmed and wild fish differ in proximate composition, colour, and especially in texture, fatty acids and free amino acids (FAAs) profiles. Flesh of wild fish was firmer, which could be attributed to their lower fat content and higher level of activity. Cultured fish showed a higher content of monounsaturated fatty acids and lower of saturated and polyunsaturated fatty acids (PUFAs). Within the PUFA group, −3 fatty acids were predominant in wild sea bass, while −6 were more abundant in farmed fish. Some FAAs related to the characteristic flavour of fish, such as glutamic acid, aspartic acid, alanine, and glycine were more abundant in cultured sea bass. No differences between fish from both farms were found, due to the similar composition of the feed used.
Cells contain a large number of antioxidants to prevent or repair the damage caused by reactive oxygen species, as well as to regulate redox-sensitive signaling pathways. General protocols are described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, whereas the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein required for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissues and cells. In general, these assays require 24-48 h to complete.
Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein - RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway.
The prospects of utilizing pretreated seawater for the culture of ( ) was evaluated under laboratory conditions with three seawater media and a control: (1) Zarrouk media (freshwater–control) (2) seawater media SW 1 (3) seawater media SW2 and (4) seawater media SW 3. The relative performance of these media were investigated with respect to their biomass production, pigment production (phycocyanin, lutein and betacarotene), and biochemical composition. . grown in media SW 2 had a biomass production (2.99 ± 0.145 g L ) comparable to that of control media (3.114 ± 0.085 g L ); highest specific growth rate (0.255 d ) and lowest doubling time (2.720 days). Phycocyanin content of the cells grown in seawater media SW 3(81.85%) was closer to that of control. Similarly the purity ratio of phycocyanin produced from cells grown in seawater media SW 3 and control were closer to 4, while the phycocyanin obtained from cells grown in other two media exhibited lower purity ratios due to accumulation of lower molecular weight carbohydrates. The phycocyanin/Chl- ratio and the betacarotene/Chl- ratio of the cells grown in seawater media were higher than control. The lutein content of cells grown in seawater media SW 2 was higher than that of control. The cells grown in seawater media had a slightly modified biochemical composition than the control with a higher carbohydrate and lower protein content. All the three seawater based media with fewer chemicals than the control (Zarrouk media) supported the growth of . as good as the control.
The in vitro production of meat is probably feasible with existing tissue engineering techniques and may offer health and environmental advantages by reducing environmental pollution and land use associated with current meat production systems. By culturing loose myosatellite cells on a substrate, it is probably possible to produce cultured meat by harvesting mature muscle cells after differentiation and processing them into various meat products. Besides reducing the animal suffering significantly, it will also ensure sustainable production of designer, chemically safe and disease free meat with favourable nutritional profile as the conditions in an in vitro meat production system are controlled and manipulatable. However, the production of highly-structured, unprocessed meat faces considerably greater technical challenges and a great deal of research is still needed to establish a sustainable in vitro meat culturing system on an industrial scale. This review discusses the requirements that need to be met to increase the feasibility of meat production in vitro, which include finding an appropriate stem cell source and being able to grow them in a three dimensional environment inside a bioreactor, providing essential cues for proliferation and differentiation.