The essential anti-angiogenic strategies in cartilage engineering and osteoarthritic cartilage repair.

In the cartilage matrix, complex interactions occur between angiogenic and anti-angiogenic components, growth factors, and environmental stressors to maintain a proper cartilage phenotype that allows for effective load bearing and force distribution. However, as seen in both degenerative disease and tissue engineering, cartilage can lose its vascular resistance. This vascularization then leads to matrix breakdown, chondrocyte apoptosis, and ossification. Research has shown that articular cartilage inflammation leads to compromised joint function and decreased clinical potential for regeneration. Unfortunately, few articles comprehensively summarize what we have learned from previous investigations. In this review, we summarize our current understanding of the factors that stabilize chondrocytes to prevent terminal differentiation and applications of these factors to rescue the cartilage phenotype during cartilage engineering and osteoarthritis treatment. Inhibiting vascularization will allow for enhanced phenotypic stability so that we are able to develop more stable implants for cartilage repair and regeneration.

Effect of Detergents on Activity and Magnesium-Dependent Properties of Different Isoforms of Na+,K+-ATPase in the Crude Membrane Fraction of Rat Cerebral Cortex.

We studied the effect of various detergents (Tween-20, Triton X-100, and sodium deoxycholate) on activity and magnesium-dependent properties of Na+,K+-ATPase of the crude membrane fraction of rat cerebral cortex. All studied detergents significantly increased activity of the studied enzyme in a concentration-dependent manner. Sodium deoxycholate provided significantly higher values Na+,K+-ATPase activity (by ≈50%) than Triton X-100 and Tween-20. In the presence of Triton X-100, a changed pattern of the dependence of enzyme activity on the concentration of magnesium ions in the incubation solution was noted. Separate measurement of activities of Na+,K+-ATPase isoforms made it possible to assume that changes in magnesium-dependent properties are due to the predominant effect of Triton X-100 on ouabain-sensitive α2- and α3-isoforms.

Integrated analytical workflow for chromatographic profiling and metabolite annotation of a cytotoxic Phorbas amaranthus extract.

Phorbas is a widely studied genus of marine sponge and produce structurally rich cytotoxic metabolites. Still, only few studies have assessed metabolites present in Brazilian species. To circumvent redundancy, in this work, we applied and herein report the use of a scouting liquid chromatographic system associate to the design of experiment produced by the DryLab® software to obtain a fast and efficient chromatographic separation of the active hexane fraction, further enabling untargeted high-resolution mass spectrometry (HRMS) data. To this end, a crude hydroalcoholic extract of the sponge Phorbas amaranthus collected in Brazilian coast was prepared and partitioned. The cytotoxicity of the crude extract and the fractions was evaluated using tumor cell culture models. Fragmentation pathways assembled from HRMS data allowed the annotation of 18 known Phorbas metabolites, while 17 metabolites were inferred based on Global Natural Product Social Molecular Networking (GNPS), matching with a further 29 metabolites annotated through molecular subnetwork. The workflow employed demonstrates that chromatographic method development can be accelerated by the use of automated scouting systems and DryLab®, which is useful for profiling natural product libraries, as well as data curation by molecular clusters and should be incorporated to the tools of natural product chemists.

Enzyme-Digested Peptides Derived from Lates calcarifer Enhance Wound Healing after Surgical Incision in a Murine Model.

Surgical wounds are common injuries of skin and tissues and usually become a clinical problem. Until now, various synthetic and natural peptides have been widely explored as potential drug candidates for wound healing. Inhibition of the TNF-α signaling pathway and promotion of angiogenesis are suggested to be involved in their effects. Angiogenesis at the wound site is one of the essential requisites for rapid healing. In the present study, a novel peptide extract derived from the natural source Lates calcarifer, commonly known as sea bass or barramundi, was evaluated for its wound healing property. The specific acidic and enzymatic approaches were employed for producing sea bass extract containing small size peptides (molecular weight ranging from 1 kD to 5 kD). The cytotoxicity of the extract was examined in HaCaT and NIH3T3. After this, the effects of enzyme digested peptide extracts of sea bass on wound healing in mice were investigated. The peptide extracts (660 and 1320 mg/kg/day) and control protein (1320 mg/kg/day) was orally given to the wounded mice, respectively, for 12 days. The surgical method was improved by implanting a silicone ring at the wound site. The ring avoided the contracting effect in murine wounds, making it more closely related to a clinical condition. The results showed promising improvement at the wound site in mice. Sea bass peptide extracts accelerated the wound healing process and enhanced the microvessel formation at the wound site. The remarkable effects of this novel sea bass peptide extract in healing traumatic injuries revealed a new option for developing wound management.

1 H NMR-based metabolomics of paired esophageal tumor tissues and serum samples identifies specific serum biomarkers for esophageal cancer.

Serum metabolites of healthy controls and esophageal cancer (EC) patients have previously been compared to predict cancer-specific profiles. However, the association between metabolic alterations in serum samples and esophageal tissues in EC patients remains unclear. Here, we analyzed 50 pairs of EC tissues and distant noncancerous tissues, together with patient-matched serum samples, using 1 H NMR spectroscopy and pattern recognition algorithms. EC patients could be differentiated from the controls based on the metabolic profiles at tissue and serum levels. Some overlapping discriminatory metabolites, including valine, alanine, glucose, acetate, citrate, succinate and glutamate, were identified in both matrices. These results suggested deregulation of metabolic pathways, and potentially revealed the links between EC and several metabolic pathways, such as the tricarboxylic acid cycle, glutaminolysis, short-chain fatty acid metabolism, lipometabolism and pyruvate metabolism. Perturbation of the pyruvate metabolism was most strongly associated with EC progression. Consequently, an optimal serum metabolite biomarker panel comprising acetate and pyruvate was developed, as these two metabolites are involved in pyruvate metabolism, and changes in their serum levels were significantly correlated with alterations in the levels of some other esophageal tissue metabolites. In comparison with individual biomarkers, this panel exhibited better diagnostic efficiency for EC, with an AUC of 0.948 in the test set, and a good predictive ability of 82.5% in the validation set. Analysis of key genes related to pyruvate metabolism in EC patients revealed patterns corresponding to the changes in serum pyruvate and acetate levels. These correlation analyses demonstrate that there were distinct metabolic characteristics and pathway aberrations in the esophageal tumor tissue and in the serum. Changes in the serum metabolic signatures could reflect the alterations in the esophageal tumor profile, thereby emphasizing the importance of distinct serum metabolic profiles as potential noninvasive biomarkers for EC.

Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography.

BACKGROUND: Maintenance of the ratio of glutathione in the reduced (GSH) and oxidised (GSSG) state in cells is important in redox control, signal transduction and gene regulation, factors that are altered in many diseases. The accurate and reliable determination of GSH and GSSG simultaneously is a useful tool for oxidative stress determination. Measurement is limited primarily to the underestimation of GSH and overestimation GSSG as a result of auto-oxidation of GSH. The aim of this study was to overcome this limitation and develop, optimise and validate a reverse-phase high performance liquid chromatographic (HPLC) assay of GSH and GSSG for the determination of oxidant status in cardiac and chronic kidney diseases. METHODS: Fluorescence detection of the derivative, glutathione-O-pthaldialdehyde (OPA) adduct was used. The assay was validated by measuring the stability of glutathione and glutathione-OPA adduct under conditions that could affect the reproducibility including reaction time and temperature. Linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), recovery and extraction efficiency and selectivity of the method were assessed. RESULTS: There was excellent linearity for GSH (r2 = 0.998) and GSSG (r2 = 0.996) over concentration ranges of 0.1 µM-4 mM and 0.2 µM-0.4 mM respectively. The extraction of GSH from tissues was consistent and precise. The limit of detection for GSH and GSSG were 0.34 µM and 0.26 µM respectively whilst their limits of quantification were 1.14 µM and 0.88 µM respectively. CONCLUSION: These data validate a method for the simultaneous measurement of GSH and GSSG in samples extracted from biological tissues and offer a simple determination of redox status in clinical samples.

Prostaglandin E2, Produced by Mast Cells in Colon Tissues From Patients With Irritable Bowel Syndrome, Contributes to Visceral Hypersensitivity in Mice.

BACKGROUND AND AIMS: Visceral hypersensitivity is common in patients with irritable bowel syndrome (IBS). We investigated whether inflammatory molecules, such as histamine and proteases, activate prostaglandin-endoperoxide synthase 2 (also called COX2) to increase the synthesis of prostaglandin E2 (PGE2) by mast cells, which activates the receptor PTGER2 (also called EP2) in the dorsal root ganglia to promote visceral hypersensitivity. METHODS: We used an enzyme-linked immunosorbent assay to measure levels of spontaneous release of molecules from mast cells in colonic mucosa from patients with IBS with diarrhea (IBS-D; 18 women and 5 men; aged 28-60 years), healthy individuals (controls, n = 24), mice, and rats. We measured visceromotor responses to colorectal distension in rodents after intracolonic administration of colon biopsy supernatants, histamine, PGE2, a small interfering RNA against EP2, or an agonist of F2R like trypsin receptor 1 (F2RL1, also called protease-activated receptor 2 [PAR2]). We investigated the role of COX2, produced by mast cells, in mediation of visceral hypersensitivity using mice with the Y385F substitution in Ptgs2 (Ptgs2Y385F mice), mast cell-deficient (W/WV) mice, and W/WV mice given injections of mast cells derived from wild-type or Ptgs2Y385F mice. RESULTS: Colon biopsies from patients with IBS-D had increased levels of PGE2, based on enzyme-linked immunosorbent assay, and COX2 messenger RNA and protein, compared with control biopsies. Immunohistochemistry showed that most of the COX2 was in mast cells. Intracolonic infusions of rats with IBS-D biopsy supernatants generated a 3- to 4-fold increase in visceromotor responses to colorectal distension; this was associated with significant increases in PGE2, histamine, and tryptase in the colonic mucosa. These increases were prevented by a mast cell stabilizer, COX2 inhibitor, or knockdown of EP2. Intracolonic administration of supernatants from biopsies of patients with IBS-D failed to induce visceral hypersensitivity or increase the level of PGE2 in W/WV and Ptgs2Y385Fmice. Reconstitution of mast cells in W/WV mice restored the visceral hypersensitivity response. CONCLUSIONS: Abnormal synthesis of PGE2 by colonic mast cells appears to induce visceral hypersensitivity in patients with IBS-D.

Metabolic changes in mice cardiac tissue after low-dose irradiation revealed by 1H NMR spectroscopy.

Ionizing radiation may cause cardiotoxicity not only at high, but even at low (considered as harmless) doses, yet the molecular mechanisms of the heart's response to low doses are not clear. In this work, we used high-resolution nuclear magnetic resonance (NMR) spectroscopy to detect the early and late effects of radiation on the metabolism of murine hearts. The hearts of C57Bl/6NCrl female mice were irradiated in vivo with single 0.2 Gy or 2 Gy doses using 6 MV photons, then tissues were collected 48 h and 20 weeks after exposure. The most distinct changes in the profile of polar metabolites were detected 48 h after irradiation with 2 Gy, and included increased levels of pantothenate and glutamate as well as decreased levels of alanine, malonate, acetylcarnitine, glycine and adenosine. Significant effects of the 2 Gy dose were also observed 20 weeks after irradiation and included decreased levels of glutamine and acetylcarnitine when compared with age-matched controls. Moreover, several differences were observed between hearts irradiated with 2 Gy and analyzed either 48 h or 20 weeks after the exposure, which included changes in levels of acetylcarnitine, alanine, glycine, glutamate, glutamine, formate, myo-inositol and trimethylamine. No statistically significant effects induced by the 0.2 Gy dose were observed 20 weeks after irradiation. In general, radiation-affected compounds were associated with energy metabolism, fatty acid beta-oxidation, oxidative stress and damage to cell structures. At the same time, radiation-related effects were not detected at the level of tissue histology, which indicated a higher sensitivity of metabolomics-based tests for cardiac tissue response to radiation.

Dysregulation of inflammatory cytokines and inhibition of VEGFA in the human umbilical cord are associated with negative pregnancy outcomes.

INTRODUCTION: Cytokines and vascular endothelial growth factors (VEGF) are involved in all aspects of pregnancy: from placentation, through fetal development, parturition and neonatal well-being. Umbilical cord inflammatory cytokines and/or VEGF have not been well studied with respect to dysregulation associated with disorders of pregnancy or maternal/neonatal outcomes. METHODS: Here we have used multiplex ELISA to screen umbilical cord lysates (comprising cord blood, endothelia and Wharton's jelly, n = 380), for levels of IFN-γ, IL1-ß, IL-6, IL-8, IL-10, TNF-α and VEGFs A, C and D and associations with 46 ICD9/10 codes encompassing obstetric, maternal and neonatal variables. RESULTS: No significant differences were observed for IFNγ, VEGFC or VEGFD with any clinical outcomes. The cytokines IL1-ß, IL-6, IL-8, IL-10, and TNF-α showed varying levels of induction and suppression with primarily fetal-placental and neonatal complications. The largest number of significant differences between umbilical cytokines and clinical outcomes were observed for chorioamnionitis (IL1-ß, IL-6, IL-8, TNF-α), and meconium passage during birth (IL1-ß, IL-6, IL-8) where significant pro-inflammatory responses occurred and sex differences in IL-8 expression were noted. In contrast, gonococcal infection showed suppressed immune response significantly lowering IL1-ß, IL-6, IL-8, IL-10 and TNF-α. For 12/46 negative pregnancy outcomes, strong suppression of VEGFA occurred. DISCUSSION: Angiogenic and inflammatory changes in the umbilical cord could be detrimental by increasing vascular permeability in the umbilical artery or vein and/or altering vascular tone, either of which would alter blood flow affecting delivery and removal of compounds. Further elucidation of inflammatory responses in the umbilical cord may provide mechanistic understanding of adverse pregnancy outcomes.

Accumulation of Advanced Glycation Endproducts and Subclinical Inflammation in Deep Tissues of Adult Patients With and Without Diabetes.

OBJECTIVES: Advanced glycation endproducts (AGEs) play a key role in the development of foot complications in people with diabetes. Skin autofluorescence (AF) might noninvasively determine tissue accumulation of AGEs. This study evaluated the association between skin AF and AGE contents in the deep tissues of those with diabetes and the further consequences of such contents. METHODS: Between September 2014 and September 2015, we studied 33 patients, with and without diabetes, who had received lower-limb amputations. Skin AF was measured. Artery, nerve and skin were harvested during surgery. AGE contents were quantified using high-performance liquid chromatography mass spectrometry and were located by immunohistochemistry staining. Inflammatory cells were also located by immunohistochemistry, immunofluorescence and scanning electron microscopy. RESULTS: Values of skin AF and AGE contents in artery, nerve and skin in patients with diabetes were higher than those in healthy patients. Skin AF was strongly affected by AGE contents in these tissues. AGE contents in various tissues were strongly correlated with each other. Differing AGEs were deposited in similar manners in the same tissues and were accompanied by inflammatory cells. CONCLUSIONS: AGE contents were strongly correlated with each other and were accompanied by inflammatory cells. Skin AF measurement could provide information about the systemic accumulation of AGEs.

Development of amide-based fluorescent probes for selective measurement of carboxylesterase 1 activity in tissue extracts.

Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered.

Burn Eschar Stimulates Fibroblast and Adipose Mesenchymal Stromal Cell Proliferation and Migration but Inhibits Endothelial Cell Sprouting.

The majority of full-thickness burn wounds heal with hypertrophic scar formation. Burn eschar most probably influences early burn wound healing, since granulation tissue only forms after escharotomy. In order to investigate the effect of burn eschar on delayed granulation tissue formation, burn wound extract (BWE) was isolated from the interface between non-viable eschar and viable tissue. The influence of BWE on the activity of endothelial cells derived from dermis and adipose tissue, dermal fibroblasts and adipose tissue-derived mesenchymal stromal cells (ASC) was determined. It was found that BWE stimulated endothelial cell inflammatory cytokine (CXCL8, IL-6 and CCL2) secretion and migration. However, BWE had no effect on endothelial cell proliferation or angiogenic sprouting. Indeed, BWE inhibited basic Fibroblast Growth Factor (bFGF) induced endothelial cell proliferation and sprouting. In contrast, BWE stimulated fibroblast and ASC proliferation and migration. No difference was observed between cells isolated from dermis or adipose tissue. The inhibitory effect of BWE on bFGF-induced endothelial proliferation and sprouting would explain why excessive granulation tissue formation is prevented in full-thickness burn wounds as long as the eschar is still present. Identifying the eschar factors responsible for this might give indications for therapeutic targets aimed at reducing hypertrophic scar formation which is initiated by excessive granulation tissue formation once eschar is removed.

Protein disulfide isomerase ameliorates ß-cell dysfunction in pancreatic islets overexpressing human islet amyloid polypeptide.

Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits in islets of type 2 diabetic patients. hIAPP misfolding and aggregation is one of the factors that may lead to ß-cell dysfunction and death. Endogenous chaperones are described to be important for the folding and functioning of proteins. Here, we examine the effect of the endoplasmic reticulum chaperone protein disulfide isomerase (PDI) on ß-cell dysfunction. Among other chaperones, PDI was found to interact with hIAPP in human islet lysates. Furthermore, intrinsically recovered PDI levels were able to restore the effect of high glucose- and palmitate-induced ß-cell dysfunction by increasing 3.9-fold the glucose-stimulated insulin secretion levels and restoring insulin content up to basal control values. Additionally, PDI transduction decreased induced apoptosis by glucolipotoxic conditions. This approach could reveal a new therapeutic target and aid in the development of strategies to improve ß-cell dysfunction in type 2 diabetic patients.

Triggering actin polymerization in Xenopus egg extracts from phosphoinositide-containing lipid bilayers.

Xenopus egg extracts are a powerful tool to reconstitute complex cell biological processes using a cell-free strategy. When used in conjunction with liposomes and supported lipid bilayers, they can recapitulate the biochemical activities occurring at the cytosol/plasma membrane interface of the cell that underlie remodeling of the actin cytoskeleton. We use these in vitro systems to elucidate how membranes and proteins collaborate to make the appropriate actin structure at a given time and place. We have recently broadened the types of membrane substrate used, and also optimized protocols for preparation of Xenopus egg extracts for actin assembly assays from membranes. Tuning the lipid composition and curvature appropriately demands an appreciation of the native phospholipid and curvature environments that can form transiently in cells. Supported lipid bilayers on glass coverslips that contain phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) make actin bundles termed filopodia-like structures that contain fascin and have vasodilator-stimulated phosphoprotein (VASP) at their growing tips, indicating that these resemble filopodia growing from the plasma membrane. The combination of PI(4,5)P2 and phosphatidylinositol 3-phosphate in curved liposomes or supported bilayers on glass nanospheres uses Snx9, Cdc42, N-WASP (neuronal-Wiskott-Aldrich syndrome protein), and Arp2/3 complex for actin polymerization suggesting that this membrane may mimic the progression from plasma membrane to endosomes. Here we describe how to prepare high-speed supernatant frog egg extracts and phosphoinositide-containing liposomes and supported lipid bilayers that can assemble actin structures. We also describe the methods we use to assay actin polymerization using microscopy and spectrofluorometry and our protocol for immunodepleting specific proteins from extracts.

Using supported bilayers to study the spatiotemporal organization of membrane-bound proteins.

Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking, and cell motility.

Reconstitution of cortical actin networks within water-in-oil emulsions.

We describe the reconstitution of dynamic cortical actin networks within cell-like compartments. The approach is based on encapsulation of Xenopus egg extract within water-in-oil emulsions. The growth of cortical actin networks is catalyzed by an amphiphilic actin nucleation-promoting factor that localizes to the water/oil interface. We first describe the preparation of cell-free Xenopus egg extract that provides all the soluble components of the actin machinery. We then describe the preparation of the amphiphilic, fluorescent actin nucleation-promoting factor that directs actin polymerization to the interface. Finally, we describe the steps required for assembly of dynamic actin cortices within water-in-oil emulsions, including the emulsification process and the sample preparation procedures. We provide recommendations for handling sensitive system components and discuss potential uses of this reconstitution approach for cytoskeletal research.

Sample collection and preparation of biofluids and extracts for gas chromatography-mass spectrometry.

To maximize the utility of gas chromatography-mass spectrometry (GC-MS) in metabonomics research, all stages of the experimental design should be standardized, including sample collection, storage, preparation, and sample separation. Moreover, the prerequisite for any GC-MS analysis is that a compound must be volatile and thermally stable if it is to be analyzed using this technique. Since many metabolites are nonvolatile and polar in nature, they are not readily amenable to analysis by GC-MS and require initial chemical derivatization of the polar functional groups in order to reduce the polarity and to increase the thermal stability and volatility of the analytes. In this chapter, an overview is presented of the optimum approach to sample collection, storage, and preparation for gas chromatography-mass spectrometry-based metabonomics with particular focus on urine samples as example of biofluids.

Gas chromatography-mass spectrometry of biofluids and extracts.

Gas chromatography-mass spectrometry (GC-MS) has been widely used in metabonomics analyses of biofluid samples. Biofluids provide a wealth of information about the metabolism of the whole body and from multiple regions of the body that can be used to study general health status and organ function. Blood serum and blood plasma, for example, can provide a comprehensive picture of the whole body, while urine can be used to monitor the function of the kidneys, and cerebrospinal fluid (CSF) will provide information about the status of the brain and central nervous system (CNS). Different methods have been developed for the extraction of metabolites from biofluids, these ranging from solvent extracts, acids, heat denaturation, and filtration. These methods vary widely in terms of efficiency of protein removal and in the number of metabolites extracted. Consequently, for all biofluid-based metabonomics studies, it is vital to optimize and standardize all steps of sample preparation, including initial extraction of metabolites. In this chapter, recommendations are made of the optimum experimental conditions for biofluid samples for GC-MS, with a particular focus on blood serum and plasma samples.

Modulating notochordal differentiation of human induced pluripotent stem cells using natural nucleus pulposus tissue matrix.

Human induced pluripotent stem cells (hiPSCs) can differentiate into notochordal cell (NC)-like cells when cultured in the presence of natural porcine nucleus pulposus (NP) tissue matrix. The method promises massive production of high-quality, functional cells to treat degenerative intervertebral discs (IVDs). Based on our previous work, we further examined the effect of cell-NP matrix contact and culture medium on the differentiation, and further assessed the functional differentiation ability of the generated NC-like. The study showed that direct contact between hiPSCs and NP matrix can promote the differentiation yield, whilst both the contact and non-contact cultures can generate functional NC-like cells. The generated NC-like cells are highly homogenous regarding the expression of notochordal marker genes. A culture medium containing a cocktail of growth factors (FGF, EGF, VEGF and IGF-1) also supported the notochordal differentiation in the presence of NP matrix. The NC-like cells showed excellent functional differentiation ability to generate NP-like tissue which was rich in aggrecan and collagen type II; and particularly, the proteoglycan to collagen content ratio was as high as 12.5-17.5 which represents a phenotype close to NP rather than hyaline cartilage. Collectively, the present study confirmed the effectiveness and flexibility of using natural NP tissue matrix to direct notochordal differentiation of hiPSCs, and the potential of using the generated NC-like cells for treating IVD degeneration.

Assessment of cytokine-modulated proteasome activity.

This chapter presents two methods for assessment of proteasome function. The first is a modification of the standard fluorogenic peptide cleavage assay which takes into account the effect of ATP on proteasome activity. This method is described in both its macro and high throughput micro-assay forms. The second is the Proteasome Constitutive Immuno-Subunit (active site) ELISA or ProCISE method. ProCISE is a modification of active site directed probe analysis and allows for convenient differentiation between active constitutive and immuno-subunits. While the utility of measuring proteasome activity and its relationship to cytokine action and inflammation are clear, the assessment and interpretation is not always straightforward. Therefore, we also discuss the pitfalls of the standard fluorogenic assay, particularly in the interpretation of results obtained, and the advantages of the newer, ProCISE assay.