Supply chain logistics – the role of the Golgi complex in extracellular matrix production and maintenance

ABSTRACT The biomechanical and biochemical properties of connective tissues are determined by the composition and quality of their extracellular matrix. This, in turn, is highly dependent on the function and organisation of the secretory pathway. The Golgi complex plays a vital role in directing matrix output by co-ordinating the post-translational modification and proteolytic processing of matrix components prior to their secretion. These modifications have broad impacts on the secretion and subsequent assembly of matrix components, as well as their function in the extracellular environment. In this Review, we highlight the role of the Golgi in the formation of an adaptable, healthy matrix, with a focus on proteoglycan and procollagen secretion as example cargoes. We then discuss the impact of Golgi dysfunction on connective tissue in the context of human disease and ageing.

Preparation of Antihypertensive Drugs in Biological Matrix with Solvent Front Position Extraction for LC–MS/MS Analysis

Solvent front position extraction procedure was used to prepare biological samples containing selected antihypertensive drugs (ramipril, lercanidipine, indapamide, valsartan, hydrochlorothiazide, perindopril, and nebivolol). Substances separated from the biological matrix components (bovine serum albumin) were quantified by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Sample preparation process was performed with the use of a prototype horizontal chamber with a moving pipette driven by a 3D printer mechanism enabling a controlled eluent flow velocity. Application of this device was advantageous for simultaneous preparation of several samples for further quantitative analysis, with a synchronized reduction of manual operations and solvent consumption. Quantitative results obtained for the majority of the investigated antihypertensive drugs in a complex biological matrix were satisfactory. The values of the %RSD were around 5% for six of the seven substances (with the exception of indapamide). The method exhibits a suitable accuracy (the relative error percentage was below 10% for most drugs). The values of LOD and LOQ were in the range of 1.19 µg/L–8.53 µg/L and 3.61 µg/L–25.8 µg/L, respectively.

Social evolution of shared biofilm matrix components

Biofilm formation is an important and ubiquitous mode of growth among bacteria. Central to the evolutionary advantage of biofilm formation is cell-cell and cell-surface adhesion achieved by a variety of factors, some of which are diffusible compounds that may operate as classical public goods - factors that are costly to produce but may benefit other cells. An outstanding question is how diffusible matrix production, in general, can be stable over evolutionary timescales. In this work, using Vibrio cholerae as a model, we show that shared diffusible biofilm matrix proteins are indeed susceptible to cheater exploitation, and that the evolutionary stability of producing these matrix components fundamentally depends on biofilm spatial structure, intrinsic sharing mechanisms of these components, and flow conditions in the environment. We further show that exploitation of diffusible adhesion proteins is localized within a well-defined spatial range around cell clusters that produce them. Based on this exploitation range and the spatial distribution of cell clusters, we construct a model of costly diffusible matrix production and relate these length scales to the relatedness coefficient in social evolution theory. Our results show that production of diffusible biofilm matrix components is evolutionarily stable under conditions consistent with natural biofilm habitats and host environments. We expect the mechanisms revealed in this study to be relevant to other secreted factors that operate as cooperative public goods in bacterial communities, and the concept of exploitation range and the associated analysis tools to be generally applicable.

Resolving the Near-Infrared Spectrum of Articular Cartilage

Objective Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining significant research interest for characterizing connective tissues, particularly articular cartilage, because there is still a largely unmet need for rapid, accurate and objective methods for assessing tissue integrity in real-time during arthroscopic surgery. This study aims to identify the NIR spectral range that is optimal for characterizing cartilage integrity by ( a) identifying the contribution of its major constituents (collagen and proteoglycans) to its overall spectrum using proxy constituent models and ( b) determining constituent-specific spectral contributions that can be used for assessment of cartilage in its physiological state. Design The NIR spectra of cartilage matrix constituent models were measured and compared with specific molecular components of organic compounds in the NIR spectral range in order to identify their bands and molecular assignments. To verify the identified bands, spectra of the model compounds were compared with those of native cartilage. Since water obscures some bands in the NIR range, spectral measurements of the native cartilage were conducted under conditions of decreasing water content to amplify features of the solid matrix components. The identified spectral bands were then compared and examined in the resulting spectra of the intact cartilage samples. Results As water was progressively eliminated from cartilage, the specific contribution of the different matrix components was observed to correspond with those identified from the proxy cartilage component models. Conclusion Spectral peaks in the regions 5500 to 6250 cm−1 and 8100 to 8600 cm−1 were identified to be effective for characterizing cartilage proteoglycan and collagen contents, respectively.

Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-β, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-β3-induced tenogenic differentiation. The MSC were incubated with and without TGF-β3 (10 ng/ml), Y-27632 (10 μM), or both. On day 1 and day 3, the signalling pathway of TGF-β and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-β3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species ( p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-β3 and ROCK inhibition were combined ( p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-β3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-β3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.

Proteoglycan Clusters as a Site of Coordinated, Multi-Dendritic Plasticity

Experience-dependent learning depends on synaptic plasticity. While plasticity in individual synapses has been extensively investigated, the mechanisms underlying coordinated changes across sets of synapses on multiple dendrites, likely needed to encode effective adaptations to a salient stimulus, are not well understood. The extracellular matrix is uniquely well suited to fulfill this function, as rapid glia-driven remodeling of its local composition powerfully impact synaptic plasticity. We show that extracellular matrix microenvironments, named CS6 clusters, dynamically form around several dendrites in response to sensory stimuli in coincidence to stimulus-driven synaptic plasticity. CS6 clusters, formed by glia-dependent secretion of extracellular matrix components surrounding sets of adjacent dendrites, may represent a novel structure supporting coordinated synaptic plasticity.