Construction of a Liver Cancer Prognostic Model Based on Interferon-Gamma-Related Genes for Revealing the Immune Landscape.

Inferferon-gamma (LFN-γ) exerts anti-tumor effects, but there is currently no reliable and comprehensive study on prognostic function of IFN-γ-related genes in liver cancer. In this study, IFN-γ-related differentially expressed genes (DEGs) in liver cancer were identified through GO/KEGG databases and open-access literature. Based on these genes, individuals with liver cancer were clustered. A prognostic model was built based on the intersection genes between differential genes in clusters and in liver cancer. Then, model predictive performance was analyzed and validated in GEO dataset. Regression analysis was fulfilled on the model, and a nomogram was utilized to evaluate model ability as an independent prognostic factor and its clinical application value. An immune-related analysis was conducted on both the H- and L-groups, with an additional investigation into link of model genes to drug sensitivity. Significant differential expression of IFN-γ-related genes was observed between the liver cancer and control groups. Subsequently, individuals with liver cancer were classified into two subtypes based on these genes, which displayed a notable difference in survival between the two subtypes. A 10-gene liver cancer prognostic model was constructed, with good prognostic performance and was an independent prognosticator for patient analysis. L-group patients possessed higher immune infiltration levels, immune checkpoint expression levels, and immunophenoscore, as well as lower TIDE scores. Drugs that had high correlations with the feature genes included SPANXB1: PF-04217903, SGX-523, MMP1: PF-04217903, DUSP13: Imatinib, TFF1: KHK-Indazole, and Fulvestrant. We built a 10-gene liver cancer prognostic model. It was found that L-group patients were more suitable for immunotherapy. This study provided valuable information on the prognosis of liver cancer.

Comprehensively prognostic and immunological analysis of VRK Serine/Threonine Kinase 1 in pan-cancer and identification in hepatocellular carcinoma.

BACKGROUND: VRK1 is a member of the vaccinia-related kinase (VRK) family of serine/threonine protein kinases, which is related to the occurrence and development of malignant tumors. The expression pattern, predictive value, and biological function of VRK1 in various cancers remain largely elusive and warrant further investigation. METHODS: Public databases, such as TCGA, GTEx, and UCEC, were utilized to comprehensively analyze the expression of VRK1 across multiple cancer types. Prognostic significance was assessed through Univariate Cox regression and Kaplan-Meier analyses. Additionally, Spearman's correlation analysis was employed to explore the potential associations between VRK1 expression and various factors, including tumor microenvironment scores, immune cell infiltration, and immune-related genes. Moreover, to validate the findings, differential expression of VRK1 in HCC tissues and cell lines was further confirmed using qPCR, Western blot, and immunohistochemistry techniques. RESULTS: The upregulation of VRK1 was observed in most cancer types, and was associated with worse prognosis in ACC, KICH, KIRP, LGG, LIHC, LUAD, MESO, and PCPG. In various cancers, VRK1 expression exhibited positive correlations with immune infiltrating cells, immune checkpoint-related genes, TMB, and MSI. Furthermore, the promoter methylation status of VRK1 varied across different tumor types, and this variation was associated with patient prognosis in certain cancers. In our experimental analyses, we observed significantly elevated expression of VRK1 in both HCC tissues and HCC cells. Functionally, we found that the downregulation of VRK1 had a profound impact on HCC cells, leading to a significant decrease in their proliferation, migration, and invasion capabilities. CONCLUSION: The expression of VRK1 exerts a notable influence on the prognosis of several tumors and exhibits a strong correlation with tumor immune infiltration. Moreover, in the context of HCC, VRK1 may act as an oncogene, actively promoting tumor progression.

Fault Diagnosis of Rotating Machinery Using Kernel Neighborhood Preserving Embedding and a Modified Sparse Bayesian Classification Model.

Fault diagnosis of rotating machinery plays an important role in modern industrial machines. In this paper, a modified sparse Bayesian classification model (i.e., Standard_SBC) is utilized to construct the fault diagnosis system of rotating machinery. The features are extracted and adopted as the input of the SBC-based fault diagnosis system, and the kernel neighborhood preserving embedding (KNPE) is proposed to fuse the features. The effectiveness of the fault diagnosis system of rotating machinery based on KNPE and Standard_SBC is validated by utilizing two case studies: rolling bearing fault diagnosis and rotating shaft fault diagnosis. Experimental results show that base on the proposed KNPE, the feature fusion method shows superior performance. The accuracy of case1 and case2 is improved from 93.96% to 99.92% and 98.67% to 99.64%, respectively. To further prove the superiority of the KNPE feature fusion method, the kernel principal component analysis (KPCA) and relevance vector machine (RVM) are utilized, respectively. This study lays the foundation for the feature fusion and fault diagnosis of rotating machinery.

Recognition of an Ala-rich C-degron by the E3 ligase Pirh2.

The ribosome-associated quality-control (RQC) pathway degrades aberrant nascent polypeptides arising from ribosome stalling during translation. In mammals, the E3 ligase Pirh2 mediates the degradation of aberrant nascent polypeptides by targeting the C-terminal polyalanine degrons (polyAla/C-degrons). Here, we present the crystal structure of Pirh2 bound to the polyAla/C-degron, which shows that the N-terminal domain and the RING domain of Pirh2 form a narrow groove encapsulating the alanine residues of the polyAla/C-degron. Affinity measurements in vitro and global protein stability assays in cells further demonstrate that Pirh2 recognizes a C-terminal A/S-X-A-A motif for substrate degradation. Taken together, our study provides the molecular basis underlying polyAla/C-degron recognition by Pirh2 and expands the substrate recognition spectrum of Pirh2.

Improved Cell-Potent and Selective Peptidomimetic Inhibitors of Protein N-Terminal Methyltransferase 1.

Protein N-terminal methyltransferase 1 (NTMT1) recognizes a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) and transfers 1-3 methyl groups to the N-terminal region of its substrates. Guided by the co-crystal structures of NTMT1 in complex with the previously reported peptidomimetic inhibitor DC113, we designed and synthesized a series of new peptidomimetic inhibitors. Through a focused optimization of DC113, we discovered a new cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM). GD562 exhibited improved inhibition of the cellular N-terminal methylation levels of both the regulator of chromosome condensation 1 and the oncoprotein SET with an IC50 value of ~50 µM in human colorectal cancer HCT116 cells. Notably, the inhibitory activity of GD562 for the SET protein increased over 6-fold compared with the previously reported cell-potent inhibitor DC541. Furthermore, GD562 also exhibited over 100-fold selectivity for NTMT1 against several other methyltransferases. Thus, this study provides a valuable probe to investigate the biological functions of NTMT1.

Discovery of a potent and dual-selective bisubstrate inhibitor for protein arginine methyltransferase 4/5.

Protein arginine methyltransferases (PRMTs) have been implicated in the progression of many diseases. Understanding substrate recognition and specificity of individual PRMT would facilitate the discovery of selective inhibitors towards future drug discovery. Herein, we reported the design and synthesis of bisubstrate analogues for PRMTs that incorporate a S-adenosylmethionine (SAM) analogue moiety and a tripeptide through an alkyl substituted guanidino group. Compound AH237 is a potent and selective inhibitor for PRMT4 and PRMT5 with a half-maximal inhibition concentration (IC50) of 2.8 and 0.42 nmol/L, respectively. Computational studies provided a plausible explanation for the high potency and selectivity of AH237 for PRMT4/5 over other 40 methyltransferases. This proof-of-principle study outlines an applicable strategy to develop potent and selective bisubstrate inhibitors for PRMTs, providing valuable probes for future structural studies.

Chemoproteomic Study Uncovers HemK2/KMT9 As a New Target for NTMT1 Bisubstrate Inhibitors.

Understanding the selectivity of methyltransferase inhibitors is important to dissecting the functions of each methyltransferase target. From this perspective, we report a chemoproteomic study to profile the selectivity of a potent protein N-terminal methyltransferase 1 (NTMT1) bisubstrate inhibitor NAH-C3-GPKK (Ki, app = 7 ± 1 nM) in endogenous proteomes. First, we describe the rational design, synthesis, and biochemical characterization of a new chemical probe 6, a biotinylated analogue of NAH-C3-GPKK. Next, we systematically analyze protein networks that may selectively interact with the biotinylated probe 6 in concert with the competitor NAH-C3-GPKK. Besides NTMT1, the designated NTMT1 bisubstrate inhibitor NAH-C3-GPKK was found to also potently inhibit a methyltransferase complex HemK2-Trm112 (also known as KMT9-Trm112), highlighting the importance of systematic selectivity profiling. Furthermore, this is the first potent inhibitor for HemK2/KMT9 reported until now. Thus, our studies lay the foundation for future efforts to develop selective inhibitors for either methyltransferase.

Structure-based Discovery of Cell-Potent Peptidomimetic Inhibitors for Protein N-Terminal Methyltransferase 1.

Protein N-terminal methyltransferases (NTMTs) catalyze the methylation of the α-N-terminal amines of proteins starting with an X-P-K/R motif. NTMT1 has been implicated in various cancers and in aging, implying its role as a potential therapeutic target. Through structural modifications of a lead NTMT1 inhibitor, BM30, we designed and synthesized a diverse set of inhibitors to probe the NTMT1 active site. The incorporation of a naphthyl group at the N-terminal region and an ortho-aminobenzoic amide at the C-terminal region of BM30 generates the top cell-potent inhibitor DC541, demonstrating increased activity on both purified NTMT1 (IC50 of 0.34 ± 0.02 µM) and the cellular α-N-terminal methylation level of regulator of chromosome condensation 1 (RCC1, IC50 value of 30 µM) in human colorectal cancer HT29 cells. Furthermore, DC541 exhibits over 300-fold selectivity to several methyltransferases. This study points out the direction for the development of more cell-potent inhibitors for NTMT1.

Spatial distribution of the microzooplankton communities in the northern South China Sea: Insights into their function in microbial food webs.

The spatial distribution of microzooplankton in the northern South China Sea was investigated in March 2016. Microzooplankton communities were dominated by cyclotrichids, aloricate oligotrichs, and choreotrichs within ciliates and the order Gymnodiniales within dinoflagellates. Microzooplankton abundance varied between 60 and 166,520 cells L-1, with higher values in the coastal diluted water, and microzooplankton biomass exhibiting a similar pattern. High densities of Akashiwo cf. sanguinea were found in the upper waters along the coast, and mixotrophs dominated the communities in all the water masses. A canonical analysis of principal coordinates showed that the spatial patterns of microzooplankton communities could be clearly discriminated in the different water masses. Our findings provide insights into the functioning of microzooplankton and the potential risk of harmful Akashiwo cf. sanguinea algal blooms in coastal waters. In addition, our study provides evidence for using microzooplankton communities as potential indicators of water masses in complex marine systems.

Selective Peptidomimetic Inhibitors of NTMT1/2: Rational Design, Synthesis, Characterization, and Crystallographic Studies.

Protein N-terminal methyltransferases (NTMTs) methylate the α-N-terminal amines of proteins starting with the canonical X-P-K/R motif. Genetic studies imply that NTMT1 regulates cell mitosis and DNA damage repair. Herein, we report the rational design and development of the first potent peptidomimetic inhibitor for NTMT1/2. Biochemical and cocrystallization studies manifest that BM30 (with a half-maximal inhibitory concentration of 0.89 ± 0.10 µM) is a competitive inhibitor to the peptide substrate and noncompetitive to the cofactor S-adenosylmethionine. BM30 exhibits over 100-fold selectivity to NTMT1/2 among a panel of 41 MTs, indicating its potential to achieve high selectivity when targeting the peptide substrate binding site of NTMT1/2. Its cell-permeable analogue DC432 (IC50 of 54 ± 4 nM) decreases the N-terminal methylation level of the regulator of chromosome condensation 1 and SET proteins in HCT116 cells. This proof-of principle study provides valuable probes for NTMT1/2 and highlights the opportunity to develop more cell-potent inhibitors to elucidate the function of NTMTs in the future.

Probing the Plasticity in the Active Site of Protein N-terminal Methyltransferase 1 Using Bisubstrate Analogues.

The bisubstrate analogue strategy is a promising approach to develop potent and selective inhibitors for protein methyltransferases. Herein, the interactions of a series of bisubstrate analogues with protein N-terminal methyltransferase 1 (NTMT1) were examined to probe the molecular properties of the active site of NTMT1. Our results indicate that a 2-C to 4-C atom linker enables its respective bisubstrate analogue to occupy both substrate- and cofactor-binding sites of NTMT1, but the bisubstrate analogue with a 5-C atom linker only interacts with the substrate-binding site and functions as a substrate. Furthermore, the 4-C atom linker is the optimal and produces the most potent inhibitor (Ki,app = 130 ± 40 pM) for NTMT1 to date, displaying more than 3000-fold selectivity for other methyltransferases and even for its homologue NTMT2. This study reveals the molecular basis for the plasticity of the active site of NTMT1. Additionally, our study outlines general guidance on the development of bisubstrate inhibitors for any methyltransferases.

Novel Propargyl-Linked Bisubstrate Analogues as Tight-Binding Inhibitors for Nicotinamide N-Methyltransferase.

Nicotinamide N-methyltransferase (NNMT) catalyzes the methyl transfer from the cofactor S-adenosylmethionine to nicotinamide and other pyridine-containing compounds. NNMT is an important regulator for nicotinamide metabolism and methylation potential. Aberrant expression levels of NNMT have been implicated in cancer, metabolic, and neurodegenerative diseases, which makes NNMT a potential therapeutic target. Therefore, potent and selective NNMT inhibitors can serve as valuable tools to investigate the roles of NNMT in its mediated diseases. Here, we applied a rational strategy to design and synthesize the tight-binding bisubstrate inhibitor LL320 through a novel propargyl linker. LL320 demonstrates a Ki value of 1.6 ± 0.3 nM, which is the most potent inhibitor to date. The cocrystal structure of LL320 confirms its interaction with both the substrate and cofactor binding sites on NNMT. Importantly, this is the first example of using the propargyl linker to construct potent methyltransferase inhibitors, which will expand our understanding of the transition state of methyl transfer.

A novel IRBF-RVM model for diagnosis of atrial fibrillation.

BACKGROUND AND OBJECTIVE: Atrial fibrillation (AF) is one of the common cardiovascular diseases, and electrocardiography (ECG) is a key indicator for the detection and diagnosis of AF and other heart diseases. In this study, an improved machine learning method is proposed for rapid modeling and accurate diagnosis of AF. METHODS: This paper presents a novel IRBF-RVM model that combines the integrated radial basis function (IRBF) and relevance vector machine (RVM), which is utilized for the diagnosis of AF. The synchronous 12-lead ECG signals are collected from the human body surface so as to fully reflect the electrical activity of the whole heart. RR intervals of the QRS-waves in ECG signals are obtained by means of the classical Pan-Tompkins algorithm. The RR-features extracted from RR intervals are adopted as the diagnostic features for AF patients. In addition, the conventional RBF-RVM model, support vector machine (SVM) and other machine learning methods are also investigated for the diagnosis of AF so as to reflect the advantage of the proposed IRBF-RVM model. The open MIT-BIH arrhythmia database (MITDB) is also used to evaluate the predictive performance of these state-of-the-art methods. RESULTS: Altogether 1056 AF patients and 904 healthy people are participated in this study and validate the effectiveness of each channel of the 12-lead ECG signals. Experimental results show that the classification rate of IRBF-RVM can reach up to 98.16% by recurring to Channel II of the 12-lead ECG signals. CONCLUSIONS: IRBF-RVM absorbs the advantages of IRBF, which makes the kernel parameter of IRBF-RVM have a much larger selectable region than RBF-RVM. In addition, RVM has faster modeling and recognition speed in comparison with SVM. This work lays the foundation for the application of RVM to accurate diagnosis of AF.

Discovery of Bisubstrate Inhibitors for Protein N-Terminal Methyltransferase 1.

Protein N-terminal methyltransferase 1 (NTMT1) plays an important role in regulating mitosis and DNA repair. Here, we describe the discovery of a potent NTMT1 bisubstrate inhibitor 4 (IC50 = 35 ± 2 nM) that exhibits greater than 100-fold selectivity against a panel of methyltransferases. We also report the first crystal structure of NTMT1 in complex with an inhibitor, which revealed that 4 occupies substrate and cofactor binding sites of NTMT1.

Design, synthesis and antithrombotic evaluation of novel non-peptide thrombin inhibitors.

Ten derivatives of 4-((1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-1-yl)methyl)benzimida-mide (I-1∼I-2, II-1∼II-8) were designed, synthesized and evaluated for their inhibitory effect on human thrombin. Compound II-7 (IC50=82.8nM), which showed the strongest thrombin inhibitory activity among the tested compounds, was chosen as the lead compound, and ten carbamate derivatives (II-9a∼II-13a, II-9b∼II-12b, II-14) were prepared and evaluated for their anticoagulant activity. The results indicate that most of the tested compounds exhibit a certain degree of inhibitory effect on thrombin-induced platelet aggregation, among which compounds II-11a (IC50=8.16µM) and II-14 (IC50=1.95µM) show better anti-platelet aggregation activity than the others. The in vivo experimental results in rat venous thrombosis model also demonstrate compounds II-11a and II-14 can significantly reduce thrombosis in a dose-response manner. It is worth pointing out that the enhanced potency of compound II-14 may be the synergetic effect of 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP) and II-7 which are generated by hydrolysis in vivo.

Design, synthesis and antithrombotic evaluation of novel dabigatran etexilate analogs, a new series of non-peptides thrombin inhibitors.

Thrombin is a serine protease that plays a key role in blood clotting, which makes it a promising target for the treatment of thrombotic diseases. Dabigatran is direct potent thrombin inhibitor. Based on bioisosteric and scaffold hopping principle, two dabigatran mimics (I-1 and II-1) in which the benzamidine moiety of dabigatran was replaced by a tricyclic fused scaffold were designed, synthesized and evaluated for their in vitro activities for inhibiting thrombin. The results reveal that compounds I-1 (IC50=9.20nM) and II-1 (IC50=7.48nM) are potent direct thrombin inhibitors and the activity is in the range of reference drug. On this basis, twenty-two ester and carbamate derivatives of I-1 or II-1 were prepared and evaluated for their anticoagulant activity. Prodrugs I-4a (IC50=0.73µM), I-4b (IC50=0.75µM), II-2a (IC50=1.44µM) and II-2b (IC50=0.91µM) display excellent effects of inhibiting thrombin induced-platelet aggregation. Moreover, compounds I-9 and II-4, which contain a cleavable moiety with anti-platelet activity, show the best anticoagulant efficacy among the tested compounds in the rat venous thrombosis model. The compounds which have better in vitro and in vivo activity were subjected to rat tail bleeding test, and the result demonstrates that compound I-9 is less likely to have bleeding risk than dabigatran etexilate.

Identification of indole-3-carboxylic acids as non-ATP-competitive Polo-like kinase 1 (Plk1) inhibitors.

A series of indole-3-carboxylic acids were designed as novel small molecular non-ATP-competitive Plk1 inhibitors. The designed compounds were synthesized and evaluated. Most of the targeted compounds showed potent Plk1 inhibitory activities and anti-proliferative characters. Particularly, 4f and 4g showed Plk1 inhibitory activity with IC50 values of 0.41 and 0.13µM, which were about 5 and 17 times more potent compared to thymoquinone, respectively. Compound 4g also showed inhibitory activity to HeLa and MCF-7 cell lines with IC50 values of 0.72 and 1.15µM, which was almost 3 and 4 times more potent than thymoquinone. Study of mechanism of action suggested that 4g was an ATP-independent and substrate-dependent Plk1 inhibitor. Moreover, 4g showed excellent Plk1 inhibitory selectivity against Plk2 and Plk3. Fluorescein isothiocyanate Annexin V/propidium iodide (PI) double-staining assay and western-blot results indicate that induction of apoptosis by 4g is involved in its anti-tumor activity. This study may provide a support for further optimization of non-ATP-competitive Plk1 inhibitors.

Design, synthesis, and evaluation of non-ATP-competitive small-molecule Polo-like kinase 1 (Plk1) inhibitors.

A series of small-molecule Plk1 inhibitors targeting the substrate-binding pocket were designed through rational drug design for the first time. The designed compounds were synthesized and their activities were evaluated in vitro. Some of the targeted compounds showed potent Plk1 inhibitory activities and anti-proliferative characters. Particularly, 5i showed Plk1 inhibitory activity with an IC50 value of 0.68 µM. Compound 5i also showed cell growth inhibitory activity on HeLa cells with an IC50 value of 0.51 µM, which is about four times more potent compared to thymoquinone. The mechanism of action suggested that 5i was an ATP-independent and substrate-dependent Plk1 inhibitor. Compound 5i demonstrated excellent Plk1 inhibitory selectivity against Plk2, Plk3, and five serine/threonine and tyrosine kinases. Our discovery and structure-activity relationship study may provide useful lead compounds for further optimization of non-ATP-competitive Plk1 inhibitors.

Loss of jab1 in osteochondral progenitor cells severely impairs embryonic limb development in mice.

The transcriptional cofactor Jab1 controls cell proliferation, apoptosis, and differentiation in diverse developmental processes by regulating the activity of various transcription factors. To determine the role of Jab1 during early limb development, we developed a novel Jab1(flox/flox) ; Prx1-Cre conditional Knockout (cKO) mutant mouse model in which Jab1 was deleted in the osteochondral progenitor cells of the limb buds. Jab1 cKO mutant mice displayed drastically shortened limbs at birth. The short-limb defect became apparent in Jab1 cKO mutants at E15.5 and increasingly worsened thereafter. By E18.5, Jab1 cKO mutant mice exhibited significantly shorter limbs with: very few hypertrophic chondrocytes, disorganized chondrocyte columns, much smaller primary ossification centers, and significantly increased apoptosis. Real-time RT-PCR analysis showed decreased expression of Sox9, Col2a1, Ihh, and Col10a1 in Jab1 cKO mutant long bones, indicating impaired chondrogenesis. Furthermore, in a micromass culture model of early limb mesenchyme cells, alcian blue staining showed a significant decrease in chondrogenesis in Jab1 cKO limb bud cells. The expression of Sox9 and its downstream targets Col2a1 and Aggrecan, as well as BMP signaling downstream targets, Noggin, Id1, and Ihh, were significantly decreased in Jab1 cKO micromass cultures. Moreover, over-expression of SOX9 in Jab1 cKO micromass cultures partially restored Col2a1and Aggrecan expression. Jab1-deficient micromass cultures also exhibited decreased BMP signaling response and reduced BMP-specific reporter activity ex vivo. In summary, our study demonstrates that Jab1 is an essential regulator of early embryonic limb development in vivo, likely in part by co-activating Sox9 and BMP signaling.

Delphinidin inhibits IL-1ß-induced activation of NF-κB by modulating the phosphorylation of IRAK-1(Ser376) in human articular chondrocytes.

OBJECTIVE: In OA, there is enhanced expression of pro-inflammatory cytokines such as IL-1ß in the affected joint. Delphinidin, an anthocyanidin found in pigmented fruits and vegetables, has been shown to possess anti-inflammatory and antioxidant properties. In the present study we determined whether delphinidin would inhibit the IL-1ß-induced activation of NF-κB in human chondrocytes and determined the mechanism of its action. METHODS: PGE2 levels and activation of NF-κB p65 in human OA chondrocytes were determined by ELISA-based assays. Protein expression of cyclo-oxygenase-2 (COX-2) and phosphorylation of kinases was determined by western immunoblotting. Expression level of mRNAs was determined by TaqMan assays. RESULTS: Delphinidin inhibited IL-1ß-induced expression of COX-2 and production of PGE2 in human chondrocytes. Delphinidin also inhibited IL-1ß-mediated phosphorylation of IL-1 receptor-associated kinase-1(Ser376), phosphorylation of IKKα/ß, expression of IKKß, degradation of IκBα, and activation and nuclear translocation of NF-κB/p65. Phosphorylation of TGF-ß-activated kinase 1 was not observed but NF-κB-inducing kinase (NIK) was phosphorylated and phosphorylation of NIK was blocked by delphinidin in IL-1ß-treated human chondrocytes. CONCLUSION: These data identify delphinidin as a novel inhibitor of IL-1ß-induced production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expression and provide new insight into the mechanism of its action. Our results also identify inhibition of IRAK1(Ser376) phosphorylation by delphinidin in IL-1ß-induced activation of NF-κB in human chondrocytes. Given the important role played by IL-1ß-induced NF-κB activation, COX-2 expression and PGE2 production in OA, our results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA.