Fibrin-fiber architecture influences cell spreading and differentiation

ABSTRACT

The mechanical and structural properties of the extracellular matrix (ECM) play an important role in regulating cell fate. The natural ECM has a complex fibrillar structure and shows nonlinear mechanical properties, which are both difficult to mimic synthetically. Therefore, systematically testing the influence of ECM properties on cellular behavior is very challenging. In this work we show two different approaches to tune the fibrillar structure and mechanical properties of fibrin hydrogels. Addition of extra thrombin before gelation increases the protein density within the fibrin fibers without significantly altering the mechanical properties of the resulting hydrogel. On the other hand, by forming a composite hydrogel with a synthetic biomimetic polyisocyanide network the protein density within the fibrin fibers decreases, and the mechanics of the composite material can be tuned by the PIC/fibrin mass ratio. The effect of the changes in gel structure and mechanics on cellular behavior are investigated, by studying human mesenchymal stem cell (hMSC) spreading and differentiation on these gels. We find that the trends observed in cell spreading and differentiation cannot be explained by the bulk mechanics of the gels, but correlate to the density of the fibrin fibers the gels are composed of. These findings strongly suggest that the microscopic properties of individual fibers in fibrous networks play an essential role in determining cell behavior.     

Surviving in Changing Seascapes: Sediment Dynamics as Bottleneck for Long-Term Seagrass Presence

Changes in the seascape often result in altered hydrodynamics that lead to coinciding changes in sediment dynamics. Little is known on how altered sediment dynamics affect long-term seagrass persistence. We studied the thresholds of sediment dynamics in relation to seagrass presence by comparing sediment characteristics and seagrass presence data of seven separate seagrass meadows. All meadows had a long-term (>20 years) presence. Within these meadows, we distinguish so-called “hotspots” (areas within a meadow where seagrass was found during all mapping campaigns) and “coldspots” (with infrequent seagrass presence). We monitored static sediment characteristics (median grain size, bulk density, silt content) and sediment dynamics (that is, bed level change and maximum sediment disturbance depth), bioturbation (that is, lugworm densities and induced fecal pit and mound relief), and seagrass cover. We statistically analyzed which sediment characteristic best explains seagrass cover. Densely vegetated hotspots were shown to have lower sediment dynamics than sparsely vegetated hotspots and coldspots, whereas static sediment characteristics were similar (grain size, bulk density). The vegetation cover was either low (2–15%) or high (>30%) and sediment dynamics showed a threshold for vegetation cover. From this correlative finding, we postulate a self-sustaining feedback of relatively dense seagrass via sediment stabilization and accordingly a runaway feedback once the seagrass cover becomes too sparse. The sensitivity for sediment dynamics shown in our study implies that future existence of seagrass meadows may be at risk as ongoing climate change might directly (increased environmental extremes) or indirectly (changing seascapes) negatively affect seagrass beds.     

Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion

Much recent work has focussed on the role of membrane-bound components in fusion. We show here that p97 and NSF are sufficient to mediate rapid membrane fusion. Fractionation of cytosol revealed that p97 and its co-factor, p47, constitutes the major fusion activity. This was confirmed by depleting p97 from the cytosol, which resulted in an 80% decrease in fusion. Using purified protein, p97 or NSF was found to be sufficient to mediate rapid fusion in an ATP-dependent manner. A regulatory role was observed for their corresponding co-factors, p47 and alpha-SNAP. When present at a molar ratio half of that of the ATPase, both co-factors increased fusion activity significantly. Intriguingly, at this ratio the ATPase activity of the complex measured in solution was at its lowest, suggesting that the co-factor stabilizes the ATP state. The fusion event involved mixing of both leaflets of the opposing membranes and contents of liposomes. We conclude from these data that p97, NSF and perhaps other related ATPases catalyse rapid and complete fusion between lipid bilayers on opposing membranes. This highlights a new role for p97 and NSF and prompts a re-evaluation of current fusion models.     

Use of capillary (zone) electrophoresis for determining felinine and it’s application to investigate the stability of felinine

Summary. A rapid capillary electrophoresis method was established to quantify felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid) in cat urine and used to investigate felinine stability. Synthetic felinine was stable in the presence of oxygen while 11% of the natural felinine in urine disappeared after 4 days exposure to air. Both synthetic felinine and the natural felinine (in urine) were stable for up to 3 months when stored at –5°C and 20°C. Thirty percent of the synthetic felinine was lost after 5 hours at 100°C while 95% of the natural felinine disappeared after only 2 hours at the same temperature. The recovery of felinine under certain conditions was greater than 100%. It is possible that acetyl-felinine may be present in the urine and that it is deacetylated during incubation. Overall synthetic felinine was found to be stable but the felinine in cat urine much less so. Other compounds present in the urine may contribute to the decomposition of felinine.     

Effects of sex and dietary phosphorus level on the apparent metabolizable energy and nutrient digestibility in broiler chickens

The influence of sex of broilers and dietary phosphorus (P) level on energy utilization and apparent ileal digestibility of N, P and phytate-P were investigated. The AME(N) was determined using 3- and 6-week old broilers, while the apparent ileal digestibility were determined only with 6-week old birds. Sex of broilers had no effect on the AME(N) values determined during week 3. During week 6, the AME(N) values for male broilers were higher (P < 0.01) than those for the females. An interaction (P < 0.05) between sex and dietary P level was also observed. AME(N) values determined with male broilers were lower in the adequate-P diet compared to those in the low-P diet, whereas no effect of P level was observed in females. Female broilers tended (P < 0.10) to have a higher ileal N digestibility than the males, but a significant (P < 0.01) sex x P level interaction was observed. Males receiving the adequate-P diet had a lower N digestibility than those receiving the low-P diet, whereas the opposite was true in the females. Ileal phytate P degradation in males was higher than in females (0.282 vs. 0.234), but the differences were not significant. Increasing dietary P level increased ileal P digestibility in both the males and females, but the improvements were greater in the females, as indicated by a significant sex x P level interaction.     

Dynamics of protein and chromophore structural changes in the photocycle of photoactive yellow protein monitored by time-resolved optical rotatory dispersion

The dynamics of the PYP photocycle have been studied using time-resolved optical rotatory dispersion (TRORD) spectroscopy in the visible and far-UV spectral regions to probe the changes in the chromophore configuration and the protein secondary structure, respectively. The changes in the secondary structure in PYP upon photoisomerization of the chromophore can be described by two exponential lifetimes of 2 +/- 0.8 and 650 +/- 100 ms that correspond to unfolding and refolding processes, respectively. The TRORD experiments that follow the dynamics of the chromophore report three exponential components, with lifetimes of 10 +/- 3 micros, 1.5 +/- 0.5 ms, and 515 +/- 110 ms. A comparison of the kinetic behaviors of the chromophore and protein shows that during the decay of pR(465) an initial relaxation that is localized to the chromophore hydrophobic pocket precedes the formation of the chromophore and protein structures found in pB(355). In contrast, the protein and chromophore processes occur with similar time constants during inactivation of the signaling state.     

The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides

The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.     

Moderate alcohol consumption: effects on lipids and cardiovascular disease risk

Light to moderate alcohol consumption is associated with a reduced risk of coronary heart disease, as well as ischaemic stroke and possibly type 2 diabetes. Epidemiological and physiological data are in favour of a causal relationship. Proposed protective mechanisms include the stimulation of HDL-mediated processes such as reverse cholesterol transport and antioxidative effects. More well-controlled studies are needed to provide a complete understanding of the complexity of the underlying physiological mechanisms.     

Allocation in recycling systems

‘Design for Recycling’ and dematerialization by enhancing the durability of products are major aspects of the quest for sustainable products. This article presents an LCA-based model for the integrated analyses of the product chain, its recycling systems, and its waste treatment systems at the ‘End of Life’ stage. The model is an extension of the EVR (Eco-costs/Value Ratio) model which has been published in this journal (Vogtländer et al. 2001), but can also be applied to other life cycle interpretation models, since the model as such is not restricted to the use of the eco-costs as a single indicator. The model has been developed to evaluate the design alternatives of complex products like buildings and cars. These products comprise several subsystems, each with its own special solution at the End of Life stage: Extending of the product life, object renovation, re-use of components, re-use of materials, useful application of waste materials, immobilization with and without useful applications, incineration with and without energy recovery, land fill. Since complex product systems always comprise a combination of these design alternatives, a methodology is given to calculate and allocate the eco-costs of the total system in order to select the best solution for sustainability. The methodology is characterized by:

1. A main allocation model of the recycling flow based on physical relationships,
2. a strict separation of the market value, the costs and the ecocosts in the system,
3. a main allocation model for extension of lifetime based on ‘depreciation of eco-costs’, parallel to economic depreciation.