Matrix Molecules Research Articles

Spatial Positioning and Matrix Programs of Cancer-Associated Fibroblasts Promote T-cell Exclusion in Human Lung Tumors.

The cellular and molecular programs driving T-cell marginalization in solid tumors remain unclear. Here, we describe two CAF populations associated with T-cell exclusion in human lung tumors. We demonstrate the importance of pairing molecular and spatial analysis of the tumor microenvironment, a prerequisite to developing new strategies targeting T cell-excluding CAF. See related commentary by Sherman, p. 2501. This article is highlighted in the In This Issue feature, p. 2483.

Characterization of Migratory Cells From Bioengineered Bovine Cartilage in a 3D Co-culture Model

Background: Chondrocyte migration in native cartilage is limited and has been implicated as one of the reasons for the poor integration of native implants. Through use of an in vitro integration model, it has previously been shown that cells from bioengineered cartilage can migrate into the native host cartilage during integration. Platelet-rich plasma (PRP) treatment further enhanced integration of bioengineered cartilage to native cartilage in vitro. However, it is not known how PRP treatment of the bioengineered construct promotes this. Hypothesis: PRP supports cell migration from bioengineered cartilage and these migratory cells have the ability to accumulate cartilage-like matrix. Study Design: Controlled laboratory study. Methods: Osteochondral-like constructs were generated by culturing primary bovine chondrocytes on the top surface of a porous bone substitute biomaterial composed of calcium polyphosphate. After 1 week in culture, the constructs were submerged in PRP and placed adjacent, but 2 mm distant, to a native bovine osteochondral plug in a co-culture model for 2 weeks. Cell migration was monitored using phase-contrast imaging. Cell phenotype was determined by evaluating the gene expression of matrix metalloprotease 13 (MMP-13), Ki67, and cartilage matrix molecules using quantitative polymerase chain reaction. When tissue formed, it was assessed by histology, immunohistochemistry, and quantification of matrix content. Results: PRP treatment resulted in the formation of a fiber network connecting the bioengineered cartilage and native osteochondral plug. Cells from both the bioengineered cartilage and the native osteochondral tissue migrated onto the PRP fibers and formed a tissue bridge after 2 weeks of culture. Migratory cells on the tissue bridge expressed higher levels of collagen types II and I (COL2, COL1), Ki67 and MMP-13 mRNA compared with nonmigratory cells in the bioengineered cartilage. Ki67 and MMP-13–positive cells were found on the edges of the tissue bridge. The tissue bridge accumulated COL1 and COL2 and aggrecan and contained comparable collagen and glycosaminoglycan content to the bioengineered cartilage matrix. The tissue bridge did not reliably develop in the absence of cells from the native osteochondral plug. Conclusion: Bioengineered cartilage formed by bovine chondrocytes contains cells that can migrate on PRP fibers and form cartilaginous tissue. Clinical Relevance: Migratory cells from bioengineered cartilage may promote cartilage integration. Further studies are required to determine the role of migratory cells in integration in vivo.

STAT3 in porcine endometrium during early pregnancy induces changes in extracellular matrix components and promotes angiogenesis†.

A molecular interaction between maternal endometrium and implanting conceptus can lead to activation of a variety of transcription factors that regulate expression of several genes necessary for the process of embryo implantation. While, signal transducer and activator of transcription 3 (STAT3) is responsible for decidualization and epithelial remodeling in humans and mice, its role in porcine endometrium has not been explored before. In the present study, we observed a pregnancy dependent increase in gene and protein expression of STAT3. Phosphorylated STAT3 was predominantly present in the endometrium of pregnant animals in luminal and glandular epithelium and in the endothelium of blood vessels with a weak staining in stromal cells. Interleukins, IL-1β and IL-6, and epidermal growth factor (EGF)-induced STAT3 expression and phosphorylation in endometrial explants collected on Day 13 of the estrous cycle. Biological significance of STAT3 was evaluated by blocking its phosphorylation with STAT3-specific inhibitor, Stattic. Using porcine extracellular matrix (ECM) and adhesion molecule array, EGF was shown to induce changes in gene expression of ECM components: MMP1, MMP3, MMP12, LAMA1, SELL, and ICAM1, which was abrogated in the presence of Stattic. Transcriptional activity of STAT3 was observed in promoter regions of MMP3 and MMP12. Additionally, IL-6-induced STAT3 phosphorylation upregulated VEGF and VCAM1 abundances in endometrial-endothelial cells (EEC). Moreover, IL-6 resulted in an increase in EEC proliferation and capillary formation which was reversed in the presence of Stattic. Results of present study reveal a role for STAT3 phosphorylation in regulating extracellular matrix remodeling and angiogenesis in porcine endometrium to facilitate embryo implantation.

Role of Perineuronal nets in the cerebellar cortex in cocaine-induced conditioned preference, extinction, and reinstatement

Perineuronal nets (PNNs) are cartilage-like structures of extracellular matrix molecules that enwrap in a net-like manner the cell-body and proximal dendrites of special subsets of neurons. PNNs stabilize their incoming connections and restrict plasticity. Consequently, they have been proposed as a candidate mechanism for drug-induced learning and memory. In the cerebellum, PNNs surround Golgi inhibitory interneurons and both inhibitory and excitatory neurons in the deep cerebellar nuclei (DCN). Previous studies from the lab showed that cocaine-induced conditioned memory increased PNN expression in the granule cell layer of the posterior vermis. The present research aimed to investigate the role of cerebellar PNNs in cocaine-induced conditioned preference. For this purpose, we use the enzyme chondroitinase ABC (ChABC) to digest PNNs at different time points of the learning process to ascertain whether their removal can affect drug-induced memory. Our results show that PNN digestion using ChABC in the posterior vermis (Lobule VIII) did not affect the acquisition of cocaine-induced conditioned preference. However, the removal of PNNs in Lobule VIII -but not in the DCN- disrupted short-term memory of conditioned preference. Moreover, although PNN digestion facilitated the formation of extinction, reinstatement of cocaine-induced conditioned preference was encouraged under PNN digestion. The present findings suggests that PNNs around Golgi interneurons are needed to maintain cocaine-induced Pavlovian memory but also to stabilize extinction memory. Conversely, PNN degradation within the DCN did not affect stability of cocaine-induced memories. Therefore, degradation of PNNs in the vermis might be used as a promising tool to manipulate drug-induced memory.

Carbonic Anhydrase-Mimicking Supramolecular Nanoassemblies for Developing Carbon Capture Membranes.

As a ubiquitous family of enzymes with high performance in converting carbon dioxide (CO2) into bicarbonate, carbonic anhydrases (CAs) sparked enormous attention for carbon capture. Nevertheless, the high cost and operational instability of CAs hamper their practical relevance, and the utility of CAs is mainly limited to aqueous applications where CO2-to-bicarbonate conversion is possible. Taking advantage of the chemical motif that endows CA-like active sites (metal-coordinated histidine), here we introduce a new line of high-performance gas separation membranes with CO2-philic behavior. We first self-assembled a histidine-based bolaamphiphile (His-Bola) molecule in the aqueous phase and coordinated the resulting entities with divalent zinc. Optimizing the supramolecular synthesis conditions ensured that the resultant nanoparticles (His-NPs) exhibit high CO2 affinity and catalytic activity. We then exploited the His-NPs as nanofillers to enhance the separation performance of Pebax MH 1657. The hydrogen-bonding interactions allowed the dispersion of His-NPs within the polymer matrix uniformly, as confirmed by microscopic, spectroscopic, and thermal analyses. The imidazole and amine functionalities of His-NPs enhanced the solubility of CO2 molecules in the polymer matrix. The CA-mimic active sites of His-NPs nanozymes, on the other hand, catalyzed the reversible hydration of CO2 molecules in humid conditions, facilitating their transport across the membranes. The resulting nanocomposite membranes displayed excellent CO2 separation performance, with a high level of stability. At a filling ratio as low as 3 wt %, we achieved a CO2 permeability of >145 Barrer and a CO2/N2 selectivity of >95 with retained performance under humid continuous gas feeds. The bio-inspired approach presented in this work offers a promising platform for designing durable and highly selective CO2 capture membranes.

Obeticholic acid aggravates liver injury by up-regulating the liver expression of osteopontin in obstructive cholestasis

AimsObeticholic acid (OCA) was approved for the treatment of primary biliary cholangitis (PBC) patients, as it can significantly improve the level of serum alkaline phosphatase. However, OCA-induced liver injury in PBC patients puts them at risk of acute chronic liver failure, thus limiting the clinical application of OCA. Osteopontin (OPN), an extracellular cell matrix molecule, is highly induced in many cholestatic liver diseases. Herein we explored whether liver injury exacerbation by OCA was related to OPN. Main methodsBile duct ligation (BDL) mice were treated with OCA (40 mg/kg) to evaluate its effect on liver injury and OPN involvement. Enzyme-linked immunosorbent assay, western blot, immunohistochemistry, and other assays were used to detect OPN levels in serum and liver. Immunohistochemistry, and immunofluorescence, among other assays, were used to evaluate the extent of ductular reaction. The extent of fibrosis was also determined using various assays, such as immunohistochemistry, quantitative real-time PCR (qPCR), and hydroxyproline assays. Key findingsOPN was overexpressed in the liver of BDL mice treated with OCA. OCA induced overexpression of OPN exacerbated ductular reaction, fibrosis, and liver inflammation, and reduced hepatocyte proliferation. SignificanceUpon liver injury, OCA upregulates the expression of OPN in the liver and accelerates disease progression. This mechanism helps explain the risk of liver damage associated with OCA.

Encapsulation of Lipase Enzyme in Silica Gel Matrix from Rice Husk Ash and Transesterification Reaction Activity Test on Palm Oil

Encapsulation is one of the immobilization methods to increase the stability of the enzyme or other biomolecules by binding or trapping the molecules in a certain matrix. Silica gel was mostly chosen as a matrix to encapsulate the enzyme due to its high thermal stability. In this research, silica gel was used to encapsulate of lipase enzyme. Silica gel was synthesized from sodium silicate by the sol-gel technique. Silica from rice husk ash was extracted using 2 M NaOH to produce a sodium silicate solution. The silica gel was synthesized using the acidification of sodium silicate solution. The various mass and volume ratios of the enzyme and sodium silicate solution were 1:1; 1:2, and 2:1 (w/v). The amount of encapsulated enzyme was analyzed with UV-Vis spectrophotometry at a wavelength of 540 nm after complexing with biuret. The encapsulated enzyme activity test was carried out for the transesterification reaction of palm oil under various conditions. Fatty Acid Methyl Esther (FAME) analysis and surface analysis of the encapsulated enzyme was performed using GC-MS and SEM-EDX, respectively. The results showed that the lipase enzyme could be well encapsulated in silica gel after aging for eight days, at a lipase and sodium silicate ratio of 1:2 (w/v) with a percentage of 93.72%, and there were still 85.76% encapsulated lipase even after being washed seven times. The transesterification reaction resulted in the highest conversion of oil to FAME (33.87%) at a mole ratio of 1:3 oil: methanol and a lipase mass of 0.5 g.

Temporal profiling of the breast tumour microenvironment reveals collagen XII as a driver of metastasis

The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.

How do the Local Physical, Biochemical, and Mechanical Properties of an Injectable Synthetic Anisotropic Hydrogel Affect Oriented Nerve Growth?

AbstractAs an injectable tissue regenerative platform, Anisogel aims to recapitulate the complex and anisotropic architecture of native extracellular matrix by the use of magneto‐responsive microgels, which are oriented under a low magnetic field of ≈100 mT, while a surrounding hydrogel matrix cross‐links around them. This system promotes the oriented growth of neurons when cultured in vitro. In this study, how the local microgel properties affect neurite outgrowth and orientation is aimed to understand using dorsal root ganglia from chicken embryos. When the surrounding matrix is a synthetic poly(ethylene glycol) hydrogel, the microgel concentration and length required to achieve oriented nerve growth is higher compared to fibrin‐based Anisogels. Microgels should be stiffer than the matrix for cells to sense the mechanical anisotropy but a wide range of microgel stiffness leads to similar cell alignment and growth. On the other hand, modification of the microgels with common extracellular matrix molecules enhances nerve growth but deteriorates nerve alignment compared to bioinert microgels in a cell adhesive surrounding gel. Finally, covalently coupling these microgels to the surrounding matrix reduces both cellular orientation and outgrowth suggesting a reduction in the ability of cells to sense the anisotropy.

Review on Molecularly Imprinted Polymer (MIP) for Electrochemical Sensor Development

Molecularly imprinted polymers (MIPs) technology has been studied extensively for multiple applications including analyte detection and chemical separation in the field of medical, pharmaceutical, food safety, and environment. Electrochemical sensors were benefitted from MIPs technology due to their chemical and physical robustness, high sensitivity, selectivity and stability, simple fabrication process, and low-cost of production. The incorporation of MIPs has allowed the development of sensors without biological elements. However, the optimization of the imprinted products requires optimal synergistic effect of multiple factors including materials selection and synthesis techniques. This optimization will form specific recognition cavities for template molecules in the polymeric matrix. This manuscript presents a summary of various MIPs synthesis techniques and performance analysis based on recent studies. The challenges faced in MIPs technology were also discussed to help future researchers in improving technology and boosting commercialization potential against the conventional electrochemical sensor.

Left ventricle- and skeletal muscle-derived fibroblasts exhibit a differential inflammatory and metabolic responsiveness to interleukin-6.

Interleukin-6 (IL-6) is an important player in chronic inflammation associated with heart failure and tumor-induced cachexia. Fibroblasts are salient mediators of both inflammation and fibrosis. Whereas the general outcome of IL-6 on the heart’s function and muscle wasting has been intensively studied, the influence of IL-6 on fibroblasts of the heart and skeletal muscle (SM) has not been analyzed so far. We illustrate that SM-derived fibroblasts exhibit higher basal mRNA expression of α-SMA, extracellular matrix molecules (collagen1a1/3a1/5a1), and chemokines (CCL2, CCL7, and CX3CL1) as compared to the left ventricle (LV)-derived fibroblasts. IL-6 drives the transdifferentiation of fibroblasts into myofibroblasts as indicated by an increase in α-SMA expression and upregulates NLRP3 inflammasome activity in both LV- and SM-derived fibroblasts. IL-6 increases the release of CCL7 to CX3CL1 in the supernatant of SM-derived fibroblasts associated with the attraction of more pro(Ly6Chi) versus anti(Ly6Clo) inflammatory monocytes as compared to unstimulated fibroblasts. IL-6-stimulated LV-derived fibroblasts attract less Ly6Chi to Ly6Clo monocytes compared to IL-6-stimulated SM-derived fibroblasts. In addition, SM-derived fibroblasts have a higher mitochondrial energy turnover and lower glycolytic activity versus LV-derived fibroblasts under basal and IL-6 conditions. In conclusion, IL-6 modulates the inflammatory and metabolic phenotype of LV- and SM-originated fibroblasts.

Brimonidine Modulates the ROCK1 Signaling Effects on Adipogenic Differentiation in 2D and 3D 3T3-L1 Cells

The additive effects of an α2-adrenergic agonist, brimonidine (BRI), on the pan-ROCK inhibitor (ROCK-i), ripasudil (Rip), and the ROCK2-I, KD025, on adipogenic differentiation (DIF+) were examined using two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells. The following analyses were carried out: (1) lipid staining (2D and 3D), (2) real-time measurements of cellular metabolism (2D), (3) mRNA expression of DIF+ related genes and extracellular matrix molecules (ECMs) including collagen (Col)-1, -4, and -6, and fibronectin (Fn), and (4) the sizes and physical properties of the 3D spheroids. The findings indicate that DIF+ induced (1) a substantial enhancement in lipid staining and enhanced expression of the Pparγ and Fabp4 genes, (2) significantly larger and softer 3D spheroids, and (3) down-regulation of Col1 and Fn and up-regulation of Col4 and Col6 genes. Treatment with Rip alone caused a significant enhancement in adipogenesis of both the 2D and 3D cultured 3T3-L1 cells and in the physical properties of the 3D spheroids; these effects were substantially inhibited by BRI, and the effects induced by BRI or KD025 were not insignificant. These collective findings indicate that the addition of BRI inhibited the Rip-induced enhancement of DIF+ in 3T3-L1 cells, presumably by modulating ROCK1 signaling.

Microbial EPS‐mediated amorphous calcium carbonate–monohydrocalcite–calcite transformations during early tufa deposition

AbstractThe Holocene Wolfenden tufa deposit in south‐eastern British Columbia, western Canada, preserves a unique record of the earliest stages of calcium carbonate deposition resulting from microbial extracellular polymeric substances‐mediated precipitation of amorphous calcium carbonate (ACC) with partial transformation to monohydrocalcite (MHC) and subsequently to nanocrystalline calcite. This is the first documentation of tufa mineralogy involving ACC transformation to MHC. Progressive dehydration triggered ACC–MHC–nanocrystalline calcite transformations on bryophytes, algae and cyanobacteria sheaths. The adsorption of extracellular polymeric substances matrix molecules into the ACC and ACC–MHC structures preserved polymorph mineralogy of incomplete transformation. Unusual concentrations of biofilm extracellular polymeric substances filaments provided nucleation sites for the ACC precipitation. The ACC nucleation calcified extracellular polymeric substances filaments and resulted in partially coalesced arrays of nanoscale ACC spheroids. Mesocrystalline structures of MHC reconfigured the concentric growth layers of ACC precipitate with bulbous ACC–MHC protuberances. Nanocrystalline rhombic faces of calcite developed within and on the surfaces of the ACC–MHC protuberances. Dehydration of these concentric growth layers of ACC–MHC resulted in the transformation into nanocrystalline calcite with substrates coalesced into micrite fabrics. Recrystallisation obliterated evidence of the calcified extracellular polymeric substances filaments and resulted in microcrystalline calcite spar domains as the widespread encrustation fabric. Localised magnesium adsorption during nucleation of the ACC within the biofilms resulted in needle calcite crystals without the precursor ACC–MHC transformation process. Microbial extracellular polymeric substances‐mediated precipitation of ACC as a necessarily critical step in the earliest phase of the tufa deposition process, leading to the nucleation of calcite has been underappreciated and generally not considered. These earliest stages of calcium carbonate precipitation are proposed as a possible template for other tufa deposits, where the evidence of microbial extracellular polymeric substances‐mediated precipitation of ACC with transformation to MHC and subsequently to nanocrystalline calcite has been obscured by recrystallisation into micrite and spar fabrics.

Structure and evolution of neuronal wiring receptors and ligands.

One of the fundamental properties of a neuronal circuit is the map of its connections. The cellular and developmental processes that allow for the growth of axons and dendrites, selection of synaptic targets, and formation of functional synapses use neuronal surface receptors and their interactions with other surface receptors, secreted ligands, and matrix molecules. Spatiotemporal regulation of the expression of these receptors and cues allows for specificity in the developmental pathways that wire stereotyped circuits. The families of molecules controlling axon guidance and synapse formation are generally conserved across animals, with some important exceptions, which have consequences for neuronal connectivity. Here, we summarize the distribution of such molecules across multiple taxa, with a focus on model organisms, evolutionary processes that led to the multitude of such molecules, and functional consequences for the diversification or loss of these receptors.

Surface Biofunctionalization of Silk Biomaterials Using Dityrosine Cross-Linking.

Biofunctionalization of silk biomaterial surfaces with extracellular matrix (ECM) molecules, cell binding peptides, or growth factors is important in a range of applications, including tissue engineering and development of implantable medical devices. Passive adsorption is the most common way to immobilize molecules of interest on preformed silk biomaterials but can lead to random molecular orientations and displacement from the surface, limiting their applications. Herein, we developed techniques for covalent immobilization of biomolecules using enzyme- or photoinitiated formation of dityrosine bonds between the molecule of interest and silk. Using recombinantly expressed domain V of the human basement membrane proteoglycan perlecan (rDV) as a model molecule, we demonstrated that rDV can be covalently immobilized via dityrosine cross-linking without the need to modify rDV or silk biomaterials. Dityrosine-based immobilization resulted in a different molecular orientation to passively absorbed rDV with less C- and N-terminal region exposure on the surface. Dityrosine-based immobilization supported functional rDV immobilization where immobilized rDV supported endothelial cell adhesion, spreading, migration, and proliferation. These results demonstrate the utility of dityrosine-based cross-linking in covalent immobilization of tyrosine-containing molecules on silk biomaterials in the absence of chemical modification, adding a simple and accessible technique to the silk biofunctionalization toolbox.

Synthesis, Characterization, and Physical Properties of Maleic Acid-Grafted Poly(butylene adipate-co-terephthalate)/Cellulose Nanocrystal Composites.

New sequences of nanocomposites including numerous maleic acid-grafted poly(butylene adipate-co-terephthalate) (g-PBAT) and cellulose nanocrystals (CNC) were efficaciously fabricated via transesterification and polycondensation processes with the covalent bonds between the polymer and reinforcing fillers. The grafting interaction of maleic acid onto PBAT was successfully demonstrated using Fourier transform infrared (FTIR) and 13C-nuclear magnetic resonance (NMR) spectra. The morphology of g-PBAT/CNC nanocomposites was investigated by wide-angle X-ray diffraction and transmission electron microscopy. Both results indicate that the CNC was randomly dispersed into the g-PBAT polymer matrix. The storage modulus at −80 and 25 °C was significantly enhanced with the incorporation of CNC into g-PBAT matrix. The crystallization rate of g-PBAT/CNC nanocomposites increased as the loading of CNC increased. With the incorporation of 3 wt% CNC, the half-time for crystallization of the g-PBAT/CNC composite decreased about 50~80% as compared with the same isothermal crystallization of pure polymer matrix. All water vapor permeation (WVP) values of all g-PBAT/CNC nanocomposites decreased as the loading of CNC increased. The decrease in WVP may be attributed to the addition of stiff CNC, causing the increase on the permeation route in the water molecules in the g-PBAT polymer matrix.

The N Terminus of Adhesion G Protein-Coupled Receptor GPR126/ADGRG6 as Allosteric Force Integrator.

The adhesion G protein–coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination or peripheral nerve repair. This renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that translates the binding of extracellular matrix (ECM) molecules to its N terminus into a metabotropic intracellular signal. To date, the structural requirements and the character of the forces needed for this ECM-mediated receptor activation are largely unknown. In this study, we provide this information by combining classic second-messenger detection with single-cell atomic force microscopy. We established a monoclonal antibody targeting the N terminus to stimulate GPR126 and compared it to the activation through its known ECM ligands, collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N terminus can be regarded as an allosteric module that can fine-tune receptor activation in a context-specific manner.

Differential Protein Expression of the Marginal Transitional Zone in Foals with Osteochondrosis

The marginal transitional zone is peripherally located within the diarthrodial joint, and represents the interface of articular cartilage, periosteum, and the fibrous joint capsule. The purpose of this study is to characterize the protein expression of matrix and molecular regulators in the marginal transitional zone of foals having osteochondrosis (OC) compared to normal foals. Several families of proteins with known roles in cartilage and bone development are investigated, including matrix molecules, Wnt signaling, apoptotic factors and paracrine cell signaling molecules. Our results demonstrate differential protein expression in the marginal transitional zone from the lateral femoral trochlear ridge of foals affected by osteochondrosis. Alterations in protein expression of OC-affected foals mainly involve components of extracellular matrix homeostasis and canonical Wnt signaling. Matrix expression of collagen type IIB and lubricin are decreased and matrix metalloproteinase-3 expression is increased in OC-affected marginal transitional zone samples. Canonical Wnt signaling is inhibited in OC-affected marginal transitional zone samples, based on increased Dickkopf-1 and decreased β-catenin protein expression. Most apoptotic and paracrine signaling proteins are not altered in OC-affected marginal transitional zone samples.

Importance of Matrix Cues on Intervertebral Disc Development, Degeneration, and Regeneration.

Back pain is one of the leading causes of disability worldwide and is frequently caused by degeneration of the intervertebral discs. The discs’ development, homeostasis, and degeneration are driven by a complex series of biochemical and physical extracellular matrix cues produced by and transmitted to native cells. Thus, understanding the roles of different cues is essential for designing effective cellular and regenerative therapies. Omics technologies have helped identify many new matrix cues; however, comparatively few matrix molecules have thus far been incorporated into tissue engineered models. These include collagen type I and type II, laminins, glycosaminoglycans, and their biomimetic analogues. Modern biofabrication techniques, such as 3D bioprinting, are also enabling the spatial patterning of matrix molecules and growth factors to direct regional effects. These techniques should now be applied to biochemically, physically, and structurally relevant disc models incorporating disc and stem cells to investigate the drivers of healthy cell phenotype and differentiation. Such research will inform the development of efficacious regenerative therapies and improved clinical outcomes.

Comprehensive Mapping and Dynamics of Site-Specific Prolyl-Hydroxylation, Lysyl-Hydroxylation and Lysyl O-Glycosylation of Collagens Deposited in ECM During Zebrafish Heart Regeneration.

Cardiac fibrosis-mediated heart failure (HF) is one of the major forms of end-stage cardiovascular diseases (CVDs). Cardiac fibrosis is an adaptive response of the myocardium upon any insult/injury. Excessive deposition of collagen molecules in the extracellular matrix (ECM) is the hallmark of fibrosis. This fibrotic response initially protects the myocardium from ventricular rupture. Although in mammals this fibrotic response progresses towards scar-tissue formation leading to HF, some fishes and urodeles have mastered the art of cardiac regeneration following injury-mediated fibrotic response. Zebrafish have a unique capability to regenerate the myocardium after post-amputation injury. Following post-amputation, the ECM of the zebrafish heart undergoes extensive remodeling and deposition of collagen. Being the most abundant protein of ECM, collagen plays important role in the assembly and cell-matrix interactions. However, the mechanism of ECM remodeling is not well understood. Collagen molecules undergo heavy post-translational modifications (PTMs) mainly hydroxylation of proline, lysine, and glycosylation of lysine during biosynthesis. The critical roles of these PTMs are emerging in several diseases, embryonic development, cell behavior regulation, and cell-matrix interactions. The site-specific identification of these collagen PTMs in zebrafish heart ECM is not known. As these highly modified peptides are not amenable to mass spectrometry (MS), the site-specific identification of these collagen PTMs is challenging. Here, we have implemented our in-house proteomics analytical pipeline to analyze two ECM proteomics datasets (PXD011627, PXD010092) of the zebrafish heart during regeneration (post-amputation). We report the first comprehensive site-specific collagen PTM map of zebrafish heart ECM. We have identified a total of 36 collagen chains (19 are reported for the first time here) harboring a total of 95 prolyl-3-hydroxylation, 108 hydroxylysine, 29 galactosyl-hydroxylysine, and 128 glucosylgalactosyl-hydroxylysine sites. Furthermore, we comprehensively map the three chains (COL1A1a, COL1A1b, and COL1A2) of collagen I, the most abundant protein in zebrafish heart ECM. We achieved more than 95% sequence coverage for all the three chains of collagen I. Our analysis also revealed the dynamics of prolyl-3-hydroxylation occupancy oscillations during heart regeneration at these sites. Moreover, quantitative site-specific analysis of lysine-O-glycosylation microheterogeneity during heart regeneration revealed a significant (p < 0.05) elevation of site-specific (K1017) glucosylgalactosyl-hydroxylysine on the col1a1a chain. Taken together, these site-specific PTM maps and the dynamic changes of site-specific collagen PTMs in ECM during heart regeneration will open up new avenues to decode ECM remodeling and may lay the foundation to tinker the cardiac regeneration process with new approaches.