Chain Precursor Research Articles

Fibrinogen alpha chain precursor and apolipoprotein A-I in urine as biomarkers for noninvasive diagnosis of calcium oxalate nephrolithiasis: a proteomics study.

Calcium oxalate nephrolithiasis is the most common urological disease, but noninvasive and convenient methods of diagnosis are rarely available. Objective. The present study aimed to identify potential urine biomarkers for noninvasive diagnosis of CaOx nephrolithiasis. Methodology. Urine samples from 72 patients with CaOx nephrolithiasis and 30 healthy controls were collected and proteomics analysis was performed using matrix-assisted laser desorption/ionization-time of flight-mass spectrometer (MALDI-TOF-MS). Results. Thirteen proteins/peptides displayed statistically significant differences. The peptides of m/z 1207.23 and 2773.86 were selected by the genetic algorithm (GA) to build a possible diagnostic model. The area under the curve of m/z 1207.23 and 2773.86 was 0.936 and 0.987, respectively. The diagnostic model in distinguishing patients and healthy subjects showed 100% sensitivity and specificity. The peak at m/z 2773.86 was identified as fibrinogen alpha chain (FGA) with the sequence G.EGDFLAEGGGVR.G, and the peak at m/z 2773.86 was identified as apolipoprotein A-I (apoA-I) with the sequence L.PVLESFKVSFLSALEEYTKKLNTQ. Conclusion. The study results strongly suggested that urinary FGA and apoA-I are highly sensitive and specific biomarkers for noninvasive diagnosis of CaOx nephrolithiasis.

Characterization of a Nagina22 rice mutant for heat tolerance and mapping of yield traits

BackgroundHeat is one of the major factors that considerably limit rice production. Nagina 22 (N22) is a deep-rooted, drought and heat tolerant aus rice cultivar. This study reports the characterization of a previously isolated dark green leaf mutant N22-H-dgl219 (NH219) which showed reduced accumulation of reactive oxygen species in leaf under 40°C heat conditions.The mutant was characterized for several traits in field under ambient (38°C) and heat stress (44°C) conditions by raising temperature artificially from flowering stage till maturity by covering plants with polythene sheets during dry season 2011. Yield traits were mapped in 70 F2 segregants of IR64 × NH219 and 36 F2 segregants of its reciprocal cross.ResultsLeaf proteome analysis using two-dimensional gel electrophoresis from N22 and NH219 showed distinct constitutive expression of ribulose bisphosphate carboxylase large chain precursor (EC 4.1.1.39) in NH219 under ambient growth condition. Heat stress resulted in reduction of all 11 traits except plant height in both N22 and NH219. The extent of reduction was more in N22 than in NH219. Both pollen viability and spikelet fertility were not reduced significantly in N22 and NH219 but reduced by 20% in IR64.ConclusionNH219 is more tolerant to heat stress than wild type N22 as its percent yield reduction is lesser than N22. Single marker analysis showed significant association of RM1089 with number of tillers and yield per plant, RM423 with leaf senescence, RM584 with leaf width and RM229 with yield per plant.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-6-36) contains supplementary material, which is available to authorized users.

Investigation of adipocyte proteome during the differentiation of brown preadipocytes

Brown adipocytes oxidize fatty acids to produce heat in response to cold or caloric overfeeding. The motivation and function of the development of brown fat may thus counteract obesity, though this remains uncertain. We investigated the brown adipocyte proteome by two-dimensional gel electrophoresis followed by mass spectrometry. Comparative analyses of proteins focused on total protein spots to filter differentially expressed proteins during the differentiation of mouse primary brown preadipocytes. A Western blot analysis was performed to verify the target proteins. The results indicated that 10 protein spots were differentially expressed with significant changes, including the three up-regulated proteins of prohibitin, hypoxanthine-guanine phosphoribosyltransferase, and enoyl-CoA hydratase protein; the 5 down-regulated proteins of triosephosphate isomerase, elongation factor 2, α-tropomyosin slow, endophilin-B1, and cofilin-1 (CFL1); and the two unequivocally expressed proteins of peroxiredoxin-1 and collagen α-1(i) chain precursor. We found that during brown adipogenesis, CFL1 has an inhibitory effect on brown adipocyte differentiation. The overexpression of CFL1 inhibited the brown fat deposition and repressed the brown marker genes UCP1, PRDM16, PGC-1α and PPARγ via actin dynamics and polymerization. These observations may be novel findings that bring new insight into the detailed mechanisms of brown adipogenesis and identify possible therapeutic targets for anti-obesity. We use 2-DE to compare the proteomes of adipocytes during the brown adipogenesis of primary mouse preadipocytes. We identified 10 proteins that are differentially expressed. Among these, we found that the actin binding protein CFL1 inhibits the differentiation of brown preadipocytes. CFL1 overexpressing cells showed lower deposition of brown fat droplets, and the brown marker genes of UCP1, PRDM16, PGC-1α and PPARγ were decreased through actin dynamics and polymerization.

The Signal Sequence Influences Post-Translational ER Translocation at Distinct Stages

The metazoan Sec61 translocon transports polypeptides into and across the membrane of the endoplasmic reticulum via two major routes, a well-established co-translational pathway and a post-translational alternative. We have used two model substrates to explore the elements of a secretory protein precursor that preferentially direct it towards a co- or post-translational pathway for ER translocation. Having first determined the capacity of precursors to enter ER derived microsomes post-translationally, we then exploited semi-permeabilized mammalian cells specifically depleted of key membrane components using siRNA to address their contribution to the membrane translocation process. These studies suggest precursor chain length is a key factor in the post-translational translocation at the mammalian ER, and identify Sec62 and Sec63 as important components acting on this route. This role for Sec62 and Sec63 is independent of the signal sequence that delivers the precursor to the ER. However, the signal sequence can influence the subsequent membrane translocation process, conferring sensitivity to a small molecule inhibitor and dictating reliance on the molecular chaperone BiP. Our data support a model where secretory protein precursors that fail to engage the signal recognition particle, for example because they are short, are delivered to the ER membrane via a distinct route that is dependent upon both Sec62 and Sec63. Although this requirement for Sec62 and Sec63 is unaffected by the specific signal sequence that delivers a precursor to the ER, this region can influence subsequent events, including both Sec61 mediated transport and the importance of BiP for membrane translocation. Taken together, our data suggest that an ER signal sequence can regulate specific aspects of Sec61 mediated membrane translocation at a stage following Sec62/Sec63 dependent ER delivery.

Potential biomarkers for adult acute myeloid leukemia minimal residual disease assessment searched by serum peptidome profiling

BackgroundPost treatment minimal residual disease (MRD) determination contributes to impending relapse prediction, chemotherapy response and clinical outcomes assessment, guiding clinicians to develop reasonable and effective individual chemotherapy options after induction/consolidation. This study was to identify serum candidate peptides for monitoring adult acute myeloid leukemia (AML) MRD.Results47 statistically different expressed peptide peaks were obtained in the molecular weight range of 700-10000 Da. Quick classifier (QC) model had optimal distinction efficiency, in the training set with a sensitivity of 90% and a specificity of 93.33%. Peptides were identified as ubiquitin-like modifier activating enzyme 1(UBA1), isoform 1 of fibrinogen alpha chain precursor and platelet factor 4(PF4). The peptide up-regulated in newly diagnosed AML patients were decreased to the normal level after CR. When refractory & relapsed, relative intensity was elevated again. Results were contrary to down-regulated peptide peaks. Western blot demonstrated that levels of the UBA1 protein did not differ between the leukemia and normal cells. Levels of isoform 1 of fibrinogen alpha chain precursor protein and PF4 protein were both decreased in leukemia cells comparing with normal cells. The serum levels of the PF4 in the newly diagnosed AML patients and healthy controls were significantly different. Further correlation analysis did not indicate the correlated relation between platelet counts and PF4 content, the correlation coefficient was 0.097. Kaplan–Meier analyses of overall survival showed that relative intensity of peptides was correlated with patient’s clinical outcome.ConclusionsWe speculate the peptides can be used as potential markers for monitoring minimal residual disease and clinical outcome assessment.

International residency for the development of the emergency department clinical nurse specialist role

Brown adipocytes oxidize fatty acids to produce heat in response to cold or caloric overfeeding. The motivation and function of the development of brown fat may thus counteract obesity, though this remains uncertain. We investigated the brown adipocyte proteome by two-dimensional gel electrophoresis followed by mass spectrometry. Comparative analyses of proteins focused on total protein spots to filter differentially expressed proteins during the differentiation of mouse primary brown preadipocytes. A Western blot analysis was performed to verify the target proteins. The results indicated that 10 protein spots were differentially expressed with significant changes, including the three up-regulated proteins of prohibitin, hypoxanthine–guanine phosphoribosyltransferase, and enoyl-CoA hydratase protein; the 5 down-regulated proteins of triosephosphate isomerase, elongation factor 2, α-tropomyosin slow, endophilin-B1, and cofilin-1 (CFL1); and the two unequivocally expressed proteins of peroxiredoxin-1 and collagen α-1(i) chain precursor. We found that during brown adipogenesis, CFL1 has an inhibitory effect on brown adipocyte differentiation. The overexpression of CFL1 inhibited the brown fat deposition and repressed the brown marker genes UCP1, PRDM16, PGC-1α and PPARγ via actin dynamics and polymerization. These observations may be novel findings that bring new insight into the detailed mechanisms of brown adipogenesis and identify possible therapeutic targets for anti-obesity.We use 2-DE to compare the proteomes of adipocytes during the brown adipogenesis of primary mouse preadipocytes. We identified 10 proteins that are differentially expressed. Among these, we found that the actin binding protein CFL1 inhibits the differentiation of brown preadipocytes. CFL1 overexpressing cells showed lower deposition of brown fat droplets, and the brown marker genes of UCP1, PRDM16, PGC-1α and PPARγ were decreased through actin dynamics and polymerization.

Unimodal and bimodal networks of physically crosslinked polyborodimethylsiloxane: viscoelastic and equibiaxial extension behaviors

Unimodal and bimodal networks of physically crosslinked polyborodimethylsiloxane (PBDMS) were prepared by end-linking hydroxy-terminated polydimethylsiloxane (PDMS) with boric acid. Their viscoelastic and equibiaxial extension behaviors were investigated. Three PDMS precursors with different number-average molecular weight (\( {\overline{M}}_n \)) were employed, of which the shortest chain had \( {\overline{M}}_n \) lower than the entanglement molecular weight. Bimodal networks were prepared from the mixture of the shortest and the longer PDMS chains. Linear viscoelastic behavior of unimodal network of the shortest chain gave the best fit to the Maxwell model with single relaxation time of 1.59 s, and equilibrium elastic modulus (Ge) of the network was well-explained by phantom network model. The unimodal networks from the other two long chain precursors, however, showed multi-relaxation behavior with the longest relaxation times of 1.00–1.26 s. Moreover, their Ge was close to affine model and deviated from the phantom model with trapped entanglement factors of ~ 0.13. The bimodal networks with high mole percentage of short chains gave Ge values approximate to the predicted values of phantom model. Such bimodal networks showed an extremely large increase in modulus at high biaxial extension, attributed by the limited extensibilities of short chains and un-relaxed crosslinked junctions.

Proteomics as a methodology for the characterization of potential new markers of stress elicited by strenuous exercise in sporting dogs – a preliminary study

Acute phase response (APR) is a well known mechanism that occurs in variety of different pathological conditions such as tissue injuries, immunological response, tumors and different inflammations. There is a lack of reports published on some stress – induced conditions in the matter of exercise or training of sporting dogs. In this study we employed the proteomic approach using 2-D gel electrophoresis combined with mass spectrometry for determination of differentially expressed proteins prior to and afterward the strenuous exercise of a German Wire-haired pointer dogs. Differentially we found that similar to serotransferrin precursor was present only after the exercise, haptoglobin heavy chain Hph, similar to fibrinogen beta chain precursor, similar to fibrinogen gamma chain precursor were up regulated after the obtained exercise unlike serum albumin precursor that was down regulated. This study has pointed out that proteomic approach has a good chance to become a reliable predictive/diagnostic tool in assessing the individualized and optimized training programme for sporting animals.

In situ polymerization and characterization of elastomeric polyurethane-cellulose nanocrystal nanocomposites. Cell response evaluation

Polyurethane/Cellulose nanocrystal (CNC) nanocomposites have been prepared by means of in situ polymerization using CNCs as precursors of polyurethane chains. Thermal, mechanical and morphological characterization has been analyzed to study the effect of CNC on the micro/nanostructure, which consisted of individualized nanocellulose crystallites covalently bonded to hard and soft segments of polyurethane. The incorporation of low CNC content led to a tough material whereas higher amount of CNC provoked an increase in soft and hard segments crystallization phenomenon. Moreover, from the viewpoint of polyurethane and polyurethane nanocomposites applications focused on biomedical devices, biocompatibility studies can be considered necessary to evaluate the influence of CNC on the biological behaviour. SEM micrographs obtained from cells seeded on top of the materials showed that L-929 fibroblasts massively colonized the materials surface giving rise to good substrates for cell adhesion and proliferation and useful as potential materials for biomedical applications.

Proteomic analysis of the biological response of MG63 osteoblast-like cells to titanium implants

Understanding of the interaction between human MG63 osteoblast-like cells and surfaces is necessary in the field of tissue engineering and biomaterials. Various titanium surfaces are widely used as not only implant materials, but also as miniscrews in orthodontics. Our goal was to assess the proteomic response of MG63 osteoblast-like cells to different titanium surfaces. MG63 osteoblast-like cells were cultured on three different titanium surfaces: a smooth surface (S), a sandblasted with large grit and acid-etched surface (SLA), and a surface coated with a thin layer of hydroxyapatite (HA). Cells grown on the rougher surfaces (SLA and HA) exhibited downregulated cell proliferation and morphological changes. In the proteomic analysis, cells grown on the SLA surface showed upregulated expression of protocadherin-β3 precursor, kinase insert domain receptor, fibroblast growth factor receptor-3, and insulin-like growth factor I, while the expression levels of cell adhesion kinase, collagen α-1(I) chain precursor, collagen type XI α2, and cadherin-11 were upregulated in cells grown on the HA surface. These proteins are known to be involved in osteoblast adhesion, growth, and differentiation. Thus, the surface properties of dental materials can influence the expression of proteins involved in osseointegration-related processes. Proteomic analysis may reveal changes in novel proteins that explain why osseointegration varies depending on surface properties.

Tumour expression of bladder cancer‐associated urinary proteins

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The current basis for diagnosis and prognosis in urinary bladder cancer is based on the pathologists' assessment of a biopsy of the tumour. Urinary biomarkers are preferable as they can be non-invasively sampled. Urinary cytology is the only test with widespread use but is hampered by poor reproducibility and low sensitivity. By studying the protein expression in bladder tumour tissue samples of proteins previously found in elevated levels in the urine of patients with bladder cancer, we have been able to show that these proteins originate from the tumour. The immunoreactivity of three of the investigated proteins increased with higher stage. Also a serine peptidase inhibitor was found to be predictive of progression from non-muscle-invasive to muscle-invasive tumours. To analyse the expression of five bladder cancer-associated urinary proteins and investigate if expression is related to the malignant phenotype of the tumour. To explore the possible prognostic value of these proteins. Urine samples, 16 from patients with bladder cancer and 26 from controls, were used in Western Blotting experiments. Tissue microarrays with bladder tissue from 344 patients diagnosed with bladder cancer between 1984 and 2005 was used in immunohistochemistry experiments. The proteins apolipoprotein E (APOE), fibrinogen β chain precursor (FGB), leucine-rich α2-glycoprotein (LRG1), polymerase (RNA) I polypeptide E (POLR1E), α1-antitrypsin (SERPINA1) and topoisomerase 2A (TOP2A) were probed with antibodies validated by the Human Protein Atlas. Increased expressions of APOE, FGB and POLR1E were correlated with increased tumour stage (P < 0.001). Expression of SERPINA1 in Ta and T1 tumours was found to increase the risk of tumour progression (hazard ratio 2.57, 95% confidence interval 1.13-5.87; P = 0.025) CONCLUSIONS: All proteins previously detected in urine from patients with bladder cancer were also expressed in bladder cancer tissue. The expression of APOE, FGB and POLR1E increased with stage and they are potential diagnostic markers. SERPINA1 was identified as a prognostic marker candidate.

Basic CuCO3/ligand as a new catalyst for 'on water' borylation of Michael acceptors, alkenes and alkynes: application to the efficient asymmetric synthesis of β‐alcohol type sitagliptin side chain

The efficient 'on water' β‐borylation using bis(pinacolato)diboron agent was achieved with a newly developed catalytic system based on basic copper carbonate and various ligands. The catalytic system was used for β‐borylation of various Michael acceptors, alkenes and alkynes. The presented methodology was successfully applied to the novel synthesis of β‐alcohol type sitagliptin side chain precursor via water‐based highly enantioselective β‐borylation followed by an oxidation process. Copyright © 2013 John Wiley &amp; Sons, Ltd.

Enantioselective Total Syntheses and Absolute Configuration of JBIR-02 and Mer-A2026B

The first total syntheses of the piericidin related natural products Mer-A2026B and JBIR-02 are reported. Key features of the synthetic approach involve an Ir-catalyzed one-pot C-H activation/oxidation procedure for the preparation of the hydroxypyridine, a vinylogous Mukaiyama aldol reaction, and a final Negishi cross-coupling of an advanced pyridine derivative with an allylic side chain precursor. In addition, the absolute configuration of Mer-A2026B (9R,10R) and JBIR-02 (9R,10R) was established.

ITIH4: A New Potential Biomarker of "Toxin Syndrome" in Coronary Heart Disease Patient Identified with Proteomic Method.

Objective. This trial aims to look for the protein biomarker of “toxin syndrome” of CHD patients. Methods. We have performed two trials in this paper. The first trial was a randomized controlled trial (RCT) of the plasma proteome in unstable angina (UA) patients by Maldi-Tof Mass. The second trial was a nested case-control study in 1503 stable CHD patients with one-year followup for acute cardiovascular events (ACEs). Results. In the RCT study, 12 protein spots were found to be the differential protein for the significant differences between the difference of before and after treatment in group A and group B; 2 of them (3207.37 Da and 4279.95 Da) was considered to be unique to “toxin syndrome” for being differential proteins of group B but not group A. These 2 spots were identified as Isoform 1 of Fibrinogen alpha chain precursor (FGA, 3207.37 Da) and Isoform 2 of inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4, 4279.95 Da), respectively. In the nested case-control study, the result of Western blot demonstrated that protein expression of ITIH4 in the group with followup ACEs was significantly lower than the matched group without followup ACEs (P = 0.027). Conclusion. ITIH4 might be a new potential biomarker of CHD “toxin syndrome” in TCM, indicating the potential role in early identifying high-risk CHD patients in stable period.

Straightforward Synthesis of a Double-Lasso Macrocycle from a Nonsymmetrical [c2]Daisy Chain

The straightforward synthesis of a double-lasso macrocycle from a nonsymmetrical [c2]daisy chain, using the copper(I)-catalyzed Huisgen alkyne-azide 1,3-dipolar cycloaddition, is described. The preparation of the nonsymmetrical alkyne azide [c2]daisy chain precursor was realized in situ via the exchange of the monomers contained in both symmetrical alkyne and azide [c2]daisy chains and was followed by mass spectrometry.

Polymer networks by molecular dynamics simulation: Formation, thermal, structural and mechanical properties

A molecular dynamics simulation method is presented and used in the study of the formation of polymer networks. We study the formation of networks representing the methylene repeating units as united atoms. The network formation is accomplished by cross-linking polymer chains with dedicated functional end groups. The simulations reveal that during the cross-linking process, initially branched molecules are formed before the gel point; approaching the gel point, larger branched entities are formed through integration of smaller branched molecules, and at the gel point a network spanning the simulation box is obtained; beyond the gel point the network continues to grow through the addition of the remaining molecules of the sol phase onto the gel (the network); the final completion of the reaction occurs by intra-network connection of dangling ends onto unsaturated cross-linkers. The conformational properties of the strands in the undeformed network are found to be very similar with the conformational properties of the chains before cross-linking. The uniaxial deformation of the formed networks is investigated and the modulus determined from the stress-strain curves shows reciprocal scaling with the precursor chain length for networks formed from sufficiently large precursor chains (N ≥ 20).

A mass spectrometry-based plasma protein panel targeting the tumor microenvironment in patients with breast cancer

Proteins secreted or shed by cancerous cells are seen as a rich source of biomarkers and novel therapeutic targets. Recently, the importance of the tumor microenvironment, which comprises the surrounding non-tumor cells, has received increased attention for its role in tumor progression. We developed a targeted proteomics assay to monitor a panel of plasma proteins postulated to be present in the tumor microenvironment. The plasma of 76 breast cancer patients was depleted of abundant circulating proteins, enzymatically digested and labeled by reductive methylation. The labeled digests were analyzed by tandem mass spectrometry using a multiple reaction monitoring acquisition method. The protein targets were correlated with the tumor characteristics, the extent of the disease and the clinical staging of the patients. Linear discriminant analysis revealed that infiltrating ductal and invasive mammary breast carcinomas could be grouped based on distinctive peptide levels of fibronectin, clusterin, gelsolin and α-1-microglobulin/Inter-α-trypsin inhibitor light chain precursor (AMBP). These proteins have been previously associated with breast cancer at the tissue level, however, this is the first study to measure plasma levels of these proteins and correlate these levels with clinical features. Significant variability was seen between unique peptides belonging to the same protein. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Evidence that a laminin-like insect protein mediates early events in the interaction of a Phytoparasite with its vector's salivary gland.

Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 β3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin β3 subunit. Anti-human laminin-5 β3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 β3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa.

Enhanced expression and fucosylation of ficolin3 in plasma of RA patients

ObjectivesThe aim of this work was to detect low abundant proteins, which may be potential biomarkers of rheumatoid arthritis (RA) at the early stage. We compared plasma protein profiles of RA patients with healthy individuals in two dimensional gel electrophoresis after removal of abundant proteins (albumin and IgG) using depletion kit and Aleuria aurantia lectin (AAL) affinity chromatography. Design and MethodsForty plasma samples each from healthy control individuals and RA patients were used in this study. ResultsWe found ficolin 3, haptoglobin alpha chain, IgM chain, alpha-1-antitrypsin and hemopexin precursor to be up regulated in the plasma of RA patients. These proteins were identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) from several reproducible 2D gels. Ficolin 3, which was not at all visible in albumin and IgG depleted gels, but detected in AAL bound fractions, was further verified by immunobloting and enzyme immunoassay. Elevated fucosylation in ficolin 3 was detected using high performance anion exchange chromatography–pulse amperometric detection (HPAEC–PAD), lectin blotting and enzyme linked lectin binding assay. ConclusionsAltered fucosylation and elevated level of Ficolin 3 might be exploited to be a potential marker for diagnosis of RA.

Osteoarthritis-related fibrosis is associated with both elevated pyridinoline cross-link formation and lysyl hydroxylase 2b expression

Fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). We investigated several factors associated with the persistence of transforming growth factor beta (TGF-β)-induced fibrosis and whether these factors also play a role in OA-related fibrosis. Mice were injected intra-articularly (i.a.) with an adenovirus encoding either TGF-β or connective tissue growth factor (CTGF). In addition, we induced OA by i.a. injection of bacterial collagenase into the right knee joint of C57BL/6 mice. mRNA was isolated from the synovium for Q-PCR analysis of the gene expression of various extracellular matrix (ECM) components, ECM degraders, growth factors and collagen cross-linking-related enzymes. Sections of murine knee joints injected with Ad-TGF-β or Ad-CTGF or from experimental OA were stained for lysyl hydroxylase 2 (LH2). The number of pyridinoline cross-links per triple helix collagen in synovium biopsies was determined with high-performance liquid chromatography (HPLC). Expression of collagen alpha-1(I) chain precursor (Col1a1), tissue inhibitor of metalloproteinases 1 (TIMP1) and especially procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2b (Plod2b) were highly upregulated by TGF-β but not by CTGF. Elevated expression of Plod2b mRNA was associated with high lysyl hydroxylase 2 (LH2) protein staining after TGF-β overexpression and in experimental OA. Furthermore, in experimental OA the number of hydroxypyridinoline cross-links was significant increased compared to control knee joints. Our data show that elevated LH2b expression is associated with the persistent nature of TGF-β-induced fibrosis. Also in experimental OA, LH2b expression as well as the number of hydroxypyridinoline cross-link were significantly upregulated. We propose that LH2b, and the subsequent increase in pyridinoline cross-links, is responsible for the persistent fibrosis in experimental OA.