Equilibrium heat-induced denaturation of chitinase 40 from Streptomyces thermoviolaceus

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (beta/alpha)8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a non-two-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, deltaG = 24.0 kcal/mol at 25 degrees C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (beta/alpha)8 barrel proteins, that their unfolding is a non-two-state process, always involving at least one unfolding intermediate.     

A 3D Hybrid Model for Tissue Growth: The Interplay between Cell Population and Mass Transport Dynamics

To provide theoretical guidance for the design and in vitro cultivation of bioartificial tissues, we have developed a multiscale computational model that can describe the complex interplay between cell population and mass transport dynamics that governs the growth of tissues in three-dimensional scaffolds. The model has three components: a transient partial differential equation for the simultaneous diffusion and consumption of a limiting nutrient; a cellular automaton describing cell migration, proliferation, and collision; and equations that quantify how the varying nutrient concentration modulates cell division and migration. The hybrid discrete-continuous model was parallelized and solved on a distributed-memory multicomputer to study how transport limitations affect tissue regeneration rates under conditions encountered in typical bioreactors. Simulation results show that the severity of transport limitations can be estimated by the magnitude of two dimensionless groups: the Thiele modulus and the Biot number. Key parameters including the initial seeding mode, cell migration speed, and the hydrodynamic conditions in the bioreactor are shown to affect not only the overall rate, but also the pattern of tissue growth. This study lays the groundwork for more comprehensive models that can handle mixed cell cultures, multiple nutrients and growth factors, and other cellular processes, such as cell death.     

Dynamic Acclimation of Photosynthesis Increases Plant Fitness in Changing Environments

Plants growing in different environments develop with different photosynthetic capacities—developmental acclimation of photosynthesis. It is also possible for fully developed leaves to change their photosynthetic capacity—dynamic acclimation. The importance of acclimation has not previously been demonstrated. Here, we show that developmental and dynamic acclimation are distinct processes. Furthermore, we demonstrate that dynamic acclimation plays an important role in increasing the fitness of plants in natural environments. Plants of Arabidopsis (Arabidopsis thaliana) were grown at low light and then transferred to high light for up to 9 d. This resulted in an increase in photosynthetic capacity of approximately 40%. A microarray analysis showed that transfer to high light resulted in a substantial but transient increase in expression of a gene, At1g61800, encoding a glucose-6-phosphate/phosphate translocator GPT2. Plants where this gene was disrupted were unable to undergo dynamic acclimation. They were, however, still able to acclimate developmentally. When grown under controlled conditions, fitness, measured as seed output and germination, was identical, regardless of GPT2 expression. Under naturally variable conditions, however, fitness was substantially reduced in plants lacking the ability to acclimate. Seed production was halved in gpt2− plants, relative to wild type, and germination of the seed produced substantially less. Dynamic acclimation of photosynthesis is thus shown to play a crucial and previously unrecognized role in determining the fitness of plants growing in changing environments.It has long been recognized that when plants are grown under a particular set of conditions they adjust their photosynthetic capacity to match those conditions (for review, see Walters, 2005). For example, early work from Bjorkman and Holmgren (1963) showed that plants of Solidago virgaurea had different photosynthetic capacities when grown either in sun or shade. In spite of its long history, however, neither the mechanism nor the significance of this response is understood (Walters, 2005). Work from Murchie and Horton (1997) showed that there is substantial variation between species in their ability to acclimate, with plants from semishaded habitats having the greatest variation in photosynthetic capacity, suggesting that there is both a benefit and cost of acclimation. Neither benefit nor cost has been demonstrated.Photosynthetic acclimation can be observed at levels ranging from whole-plant morphology to the detailed stoichiometry of the photosynthetic apparatus (Boardman, 1977; Walters, 2005). Plants grown at low light tend to invest more in leaves than in roots and to have thinner leaves. They have more chlorophyll-containing light-harvesting proteins relative to light-using enzymes involved in electron transport and metabolism, meaning that photosynthesis saturates with light at a lower irradiance. Plants can also adjust the relative proportions of the different photosystems to suit the light quality they experience (Chow et al., 1990; Walters and Horton, 1995a, 1995b).Most studies that have examined the acclimation of plants have done so by making measurements on material that has experienced only one set of conditions—e.g. either high or low light. Differences between plants therefore reflect the conditions experienced as the leaves develop, with leaf morphology and composition being optimized for the conditions seen. Plants do not, however, exist in static environments. Even for a plant growing in an unshaded location, the light incident on a leaf can vary by an order of magnitude from second to second, day to day, and week to week depending on the weather conditions. This variation will typically be accompanied by variation in the temperature, which will also impact on metabolic capacity.When plants are exposed to light at irradiances that are above saturating for photosynthesis, which may result from increases in light or from environmental conditions (e.g. cold, drought) restricting metabolism, they are liable to suffer from stress (Demmig-Adams and Adams, 1992). Specifically, excess light can give rise to reactive oxygen species (Asada, 2006). The damaging effects of this can be limited by investing in antioxidant systems; however, these are metabolically expensive with, for example, substantial amounts of individual antioxidants such as ascorbic acid, being found in the chloroplast (Asada, 2006). It seems likely therefore that the ability of a plant to minimize stress, by adjusting photosynthetic capacity to suit as well as possible the prevailing conditions, will benefit the plant and increase overall fitness.In this study, we have investigated photosynthetic acclimation of the model plant Arabidopsis (Arabidopsis thaliana). Starting with a microarray analysis, we have identified a gene that is essential for acclimation to increases in irradiance. We further show that the ability to acclimate to changes in light has a major role in determining fitness under naturally variable light conditions.     

MapQuant: open-source software for large-scale protein quantification

Whole-cell protein quantification using MS has proven to be a challenging task. Detection efficiency varies significantly from peptide to peptide, molecular identities are not evident a priori, and peptides are dispersed unevenly throughout the multidimensional data space. To overcome these challenges we developed an open-source software package, MapQuant, to quantify comprehensively organic species detected in large MS datasets. MapQuant treats an LC/MS experiment as an image and utilizes standard image processing techniques to perform noise filtering, watershed segmentation, peak finding, peak fitting, peak clustering, charge-state determination and carbon-content estimation. MapQuant reports abundance values that respond linearly with the amount of sample analyzed on both low- and high-resolution instruments (over a 1000-fold dynamic range). Background noise added to a sample, either as a medium-complexity peptide mixture or as a high-complexity trypsinized proteome, exerts negligible effects on the abundance values reported by MapQuant and with coefficients of variance comparable to other methods. Finally, MapQuant's ability to define accurate mass and retention time features of isotopic clusters on a high-resolution mass spectrometer can increase protein sequence coverage by assigning sequence identities to observed isotopic clusters without corresponding MS/MS data.     

Use of staurosporine, an actin-modifying agent, to enhance fibrochondrocyte matrix gene expression and synthesis

Modulation of the actin cytoskeleton in chondrocytes has been used to prevent or reverse dedifferentiation and to enhance protein synthesis. We have hypothesized that an actin-modifying agent, staurosporine, could be used with fibrochondrocytes to increase the gene expression and synthesis of critical fibrocartilage proteins. A range of concentrations (0.1–100 nM) was applied to fibrochondrocytes in monolayer and evaluated after 24 h and after 4 days. High-dose staurosporine treatment (10–100 nM) increased cartilage oligomeric matrix protein 60– to 500-fold and aggrecan gene expression two-fold. This effective range of staurosporine was then applied to scaffoldless tissue-engineered fibrochondrocyte constructs for 4 weeks. Whereas glycosaminoglycan synthesis was not affected, collagen content doubled, from 27.6 ± 8.8 μg in the untreated constructs to 55.2 ± 12.2 μg per construct with 100 nM treatment. When analyzed for specific collagens, the 10-nM group showed a significant increase in collagen type I content, whereas collagen type II was unaffected. A concomitant dose-dependent reduction was noted in construct contraction, reflecting the actin-disrupting action of staurosporine. Thus, staurosporine increases the gene expression for important matrix proteins and can be used to enhance matrix production and reduce contraction in tissue-engineered fibrocartilage constructs. The authors gratefully acknowledge NIAMS R01 AR 47839–2 for funding this work, and the Hertz Foundation for their support of G. Hoben.     

Inducing articular cartilage phenotype in costochondral cells

Introduction

Costochondral cells may be isolated with minimal donor site morbidity and are unaffected by pathologies of the diarthrodial joints. Identification of optimal exogenous stimuli will allow abundant and robust hyaline articular cartilage to be formed from this cell source.

Methods

In a three factor, two level full factorial design, the effects of hydrostatic pressure (HP), transforming growth factor β1 (TGF-β1), and chondroitinase ABC (C-ABC), and all resulting combinations, were assessed in third passage expanded, redifferentiated costochondral cells. After 4 wks, the new cartilage was assessed for matrix content, superficial zone protein (SZP), and mechanical properties.

Results

Hyaline articular cartilage was generated, demonstrating the presence of type II collagen and SZP, and the absence of type I collagen. TGF-β1 upregulated collagen synthesis by 175% and glycosaminoglycan synthesis by 75%, resulting in a nearly 200% increase in tensile and compressive moduli. C-ABC significantly increased collagen content, and fibril density and diameter, leading to a 125% increase in tensile modulus. Hydrostatic pressure increased fibril diameter by 30% and tensile modulus by 45%. Combining TGF-β1 with C-ABC synergistically increased collagen content by 300% and tensile strength by 320%, over control. No significant differences were observed between C-ABC/TGF-β1 dual treatment and HP/C-ABC/TGF-β1.

Conclusions

Employing biochemical, biophysical, and mechanical stimuli generated robust hyaline articular cartilage with a tensile modulus of 2 MPa and a compressive instantaneous modulus of 650 kPa. Using expanded, redifferentiated costochondral cells in the self-assembling process allows for recapitulation of robust mechanical properties, and induced SZP expression, key characteristics of functional articular cartilage.     

Temporal assessment of ribose treatment on self-assembled articular cartilage constructs

Articular cartilage cannot repair itself in response to degradation from injury or osteoarthritis. As such, there is a substantial clinical need for replacements of damaged cartilage. Tissue engineering aims to fulfill this need by developing replacement tissues in vitro. A major goal of cartilage tissue engineering is to produce tissues with robust biochemical and biomechanical properties. One technique that has been proposed to improve these properties in engineered tissue is the use of non-enzymatic glycation to induce collagen crosslinking, an attractive solution that may avoid the risks of cytotoxicity posed by conventional crosslinking agents such as glutaraldehyde. The objectives of this study were (1) to determine whether continuous application of ribose would enhance biochemical and biomechanical properties of self-assembled articular cartilage constructs, and (2) to identify an optimal time window for continuous ribose treatment. Self-assembled constructs were grown for 4 weeks using a previously established method and were subjected to continuous 7-day treatment with 30 mM ribose during culture weeks 1, 2, 3, or 4, or for the entire 4-week culture. Control constructs were grown in parallel, and all groups were evaluated for gross morphology, histology, cellularity, collagen and sulfated glycosaminoglycan (GAG) content, and compressive and tensile mechanical properties. Compared to control constructs, it was found that treatment with ribose during week 2 and for the entire duration of culture resulted in significant 62% and 40% increases in compressive stiffness, respectively; significant 66% and 44% increases in tensile stiffness; and significant 50% and 126% increases in tensile strength. Similar statistically significant trends were observed for collagen and GAG. In contrast, constructs treated with ribose during week 1 had poorer biochemical and biomechanical properties, although they were significantly larger and more cellular than all other groups. We conclude that non-enzymatic glycation with ribose is an effective method for improving tissue engineered cartilage and that specific temporal intervention windows exist to achieve optimal functional properties.     

Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD)

Osteoarthritis, a degenerative disease of the load-bearing joints, greatly reduces quality of life for millions of Americans and places a tremendous cost on the American healthcare system. Due to limitations of current treatments, tissue engineering of articular cartilage may provide a promising therapeutic option to treat cartilage defects. However, cartilage tissue engineering has yet to recapitulate the functional properties of native tissue. During normal joint loading, cartilage tissue experiences variations in osmolarity and subsequent changes in ionic concentrations. Motivated by these known variations in the cellular microenvironment, this study sought to improve the mechanical properties of neocartilage constructs via the application of hyperosmolarity and transient receptor potential vanilloid 4 (TRPV4) channel activator 4α-phorbol 12,13-didecanoate (4αPDD). It was shown that 4αPDD elicited significant increases in compressive properties. Importantly, when combined, 4αPDD positively interacted with hyperosmolarity to modulate its effects on tensile stiffness and collagen content. Thus, this study supports 4αPDD-activated channel TRPV4 as a purported mechanosensor and osmosensor that can facilitate the cell and tissue level responses to improve the mechanical properties of engineered cartilage. To our knowledge, this study is the first to systematically evaluate the roles of hyperosmolarity and 4αPDD on the functional (i.e., mechanical and biochemical) properties of self-assembled neotissue. Future work may combine 4αPDD-induced channel activation with other chemical and mechanical stimuli to create robust neocartilages suitable for treatment of articular cartilage defects.     

Cartilage Tissue Engineering Using Dermis Isolated Adult Stem Cells: The Use of Hypoxia during Expansion versus Chondrogenic Differentiation

Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O₂) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O₂) or normoxic (20% O₂) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p<0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p<0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering.     

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.