The impact of the digital economy on industrial structure upgrading in resource-based cities: Evidence from China.

The digital economy provides a new path to promote industrial structure upgrading. Using panel data from 2011 to 2020 for 85 resource-based cities in China, this paper empirically investigates the impact of the digital economy on industrial structure upgrading and the primary mechanism. The results show that the digital economy is conducive to promoting industrial structure upgrading in resource-based cities, and innovation is the primary mechanism of action. According to the different stages of resource development, we classify resource-based cities into growth, maturity, decline, and regeneration cities, and we further analyze the heterogeneous influence. In terms of influence degree, the digital economy has a more prominent role in promoting industrial structure upgrading in resource-exhausted cities. In addition, we also found that the closer to the provincial capital city, the more pronounced the promotion of the digital economy to the industrial structure upgrading.

Catalpolaglycone disrupts mitochondrial thermogenesis by specifically binding to a conserved lysine residue of UCP2 on the proton leak tunnel.

BACKGROUND: Catalpol (CAT), a naturally occurring iridoid glycoside sourced from the root of Rehmannia glutinosa, affects mitochondrial metabolic functions. However, the mechanism of action of CAT against pyrexia and its plausible targets remain to be fully elucidated. PURPOSE: This study aimed to identify the specific targets of CAT for blocking mitochondrial thermogenesis and to unveil the unique biological mechanism of action of the orthogonal binding mode between the hemiacetal group and lysine residue on the target protein in vivo. METHODS: Lipopolysaccharide (LPS)/ carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-induced fever models were established to evaluate the potential antipyretic effects of CAT. An alkenyl-modified CAT probe was designed to identify and capture potential targets. Binding capacity was tested using in-gel imaging and a cellular thermal shift assay. The underlying antipyretic mechanisms were explored using biochemical and molecular biological methods. Catalpolaglycone (CA) was coupled with protein profile identification and molecular docking analysis to evaluate and identify its binding mode to UCP2. RESULTS: After deglycation of CAT in vivo, the hemiacetal group in CA covalently binds to Lys239 of UCP2 in the mitochondria of the liver via an ɛ-amine nucleophilic addition. This irreversible binding affects proton leakage and improves mitochondrial membrane potential and ADP/ATP transformation efficiency, leading to an antipyretic effect. CONCLUSION: Our findings highlight the potential role of CA in modulating UCP2 activity or function within the mitochondria and open new avenues for investigating the therapeutic effects of CA on mitochondrial homeostasis.

Network meta-analysis of first-line thrombectomy strategy for acute posterior circulation strokes: a preliminary evaluation for combined approach.

Objective: Thrombectomy may provide superior results compared to best medical care for acute posterior circulation strokes (PCS). Contact aspiration (CA), stent retriever (SR), and combined SR + CA (SRA) are commonly employed as first-line techniques. However, the optimal strategy and the role of SRA remain uncertain. Methods: Systematic searching was conducted in three databases (PubMed, Embase, and Cochrane). Network meta-analyzes were performed using random-effects models. The reperfusion and clinical outcomes were compared. Pooled outcomes were presented as odds ratios (OR) with 95% confidence intervals (CI). Rankograms with surface under the cumulative ranking curve (SUCRA) were calculated. Results: Seventeen studies were included, involving a total of 645 patients who received first-line CA, 850 patients who received SR, and 166 patients who received SRA. Regarding final recanalization outcomes, both first-line SRA (OR = 3.2, 95%CI 1.4-11.0) and CA (OR = 2.1, 95%CI 1.3-3.7) demonstrated superiority over SR in achieving successful reperfusion [modified Thrombolysis In Cerebral Infarction (mTICI) 2b-3], with values of SUCRA 91.1, 58.5, and 0.4%, respectively. In addition, first-line SRA showed an advantage in achieving final mTICI 2c/3 compared to CA (OR = 3.6, 95%CI 0.99-16.0) and SR (OR = 6.4, 95%CI 1.3-35.0), with SUCRA value of 98.0, 44.7, and 7.2%, respectively. Regarding reperfusion outcome after the first pass, SRA also achieved a higher rate of mTICI 3 than SR (OR = 4.1, 95%CI 1.3-14.0), while CA did not (SUCRA 97.4, 4.6, 48.0%). In terms of safety outcomes, first-line CA was associated with a lower incidence of symptomatic intracranial hemorrhage (sICH) compared to SR (OR = 0.38, 95%CI 0.1-1.0), whereas the SRA technique did not (SUCRA 15.6, 78.6, 55.9%). Regarding clinical prognosis, first-line CA achieved a higher proportion of functional independence (modified Rankin Scale (mRS) 0-2) at 90 days than SR (OR = 1.4, 95%CI 1.1-1.9), whereas SRA did not (SUCRA 90.5, 17.4, 42.1%). Conclusion: For acute PCS, a first-line CA strategy yielded better results in terms of final successful reperfusion and 90-day functional independence compared to SR. As the combined technique, first-line SRA was associated with superior first-pass and final reperfusion outcomes compared to SR. However, no significant difference was observed in functional independence achieved by first-line SRA compared to the other two strategies. Further high-quality studies are warranted.

Serum Sestrin2 Emerges as a Prognostic Biomarker of Human Aneurysmal Subarachnoid Hemorrhage: A Prospective Observational Cohort Single-Center Study.

Background: Sestrin2 functions as a neuroprotective factor. Herein, serum sestrin2 was investigated with respect to its associations with severity, delayed cerebral ischemia (DCI) and prognosis of aneurysmal subarachnoid hemorrhage (aSAH). Methods: In this prospective, observational, cohort, single-center study, serum sestrin2 levels were measured at entry into the study in 45 healthy controls and at admission in 135 aSAH patients. Also, they were gauged in other time points (namely, at days 1, 2, 3, 5 and 7) among 45 patients. Unfavorable prognosis was defined as extended Glasgow Outcome Scale (GOSE) scores of 1-4 at six months after aSAH. Results: Serum sestrin2 levels were immediately raised at admission in patients, increased thereafter, peaked at day 2, declined afterwards till day 7, and were significantly higher than those in controls (all P<0.001). Serum sestrin2 levels had independent correlation with Hunt-Hess scores (beta, 1.715; 95% confidence interval (CI), 0.595-2.835; P=0.003) and modified Fisher scores (beta, 2.505; 95% CI, 1.102-3.907; P=0.001). Alternatively, serum sestrin2 levels, which were independently correlated with 6-month GOSE scores (beta, -0.050; 95% CI, -0.099-0.001; P=0.044), were independently associated with DCI (odds ratio, 1.079; 95% CI, 1.008-1.156; P=0.029) and unfavorable prognosis (odds ratio, 1.093; 95% CI, 1.020-1.172; P=0.012). DCI and prognosis prediction models, which were composed of serum sestrin2, Hunt-Hess scores and modified Fisher scores, were comparatively stable and clinically beneficial under calibration curve and decision curve. Prognosis prediction model showed significantly higher area under receiver operating characteristic curve than serum sestrin2, Hunt-Hess scores and modified Fisher scores alone (all P<0.05). Conclusion: A significant enhancement of serum sestrin2 levels after aSAH is independently related to severity, DCI and poor prognosis following aSAH. The models incorporating serum sestrin2 perform well in predicting the DCI and prognosis of aSAH patients. Presumably, determination of serum sestrin2 may be of clinical significance in aSAH.

Fuziline Ameliorates Glucose and Lipid Metabolism by Activating Beta Adrenergic Receptors to Stimulate Thermogenesis.

Radix aconiti carmichaeli is a widely used traditional Chinese medicine that has been found to be effective in treating cardiovascular diseases and metabolic disorders. Patients with these diseases often experience a heat generation disorder, which is characterized by chilliness and can worsen the progression of the disease. This study established an in vitro screening model combining the examination of cellular mitochondrial membrane potential and mitochondrial temperature to screen drugs with thermogenic activity. After differentiation and determination of the content of characteristic metabolites of the drug-containing serum blood components, it was found that Fuziline (FZL) is the key thermogenic property in Radix aconiti carmichaeli, responsible for its thermogenic effects with a high relative importance of 33%. Experiments were conducted to evaluate the thermogenic activity of Radix aconiti carmichaeli and FZL in vivo by assessing temperature changes in various organs, including the rectum, liver, and brown adipose tissue. Moreover, the effects of intracellular ß3-adrenergic receptor (ß3-AR) agonistic effects were evaluated using transient ß3-AR transfection and dual-luciferase assay systems. The molecular mechanism by which FZL promotes thermogenesis and improves mitochondrial function was investigated by verifying the ß-adrenergic receptors (ß-AR) downstream signaling pathway. The results suggest that FZL activates ß-AR nonselectively, which in turn activates the downstream cAMP-PKA signaling pathway and leads to an increase in liver glycogenolysis and triglyceride hydrolysis, accompanied by enhancing mitochondrial energy metabolism. Consequently, the liver and brown adipose tissue receive energy to generate heat. In summary, these findings provide insight into the therapeutic application of Radix aconiti carmichaeli for metabolic disorders associated with heat generation disorders.

Myeloid-derived growth factor regulates high glucose-mediated apoptosis of gingival fibroblasts and induce AKT pathway activation and nuclear factor κB pathway inhibition.

Background: /purpose: Periodontal disease is a chronic inflammatory disease that occurs in the tissues that support and attach teeth. There is considerable evidence of a relationship between diabetes and periodontal disease. Emerging studies have reported that myeloid-derived growth factor (MYDGF) can inhibit apoptosis and inflammation. The purpose of this study was to investigate whether MYDGF mediates the role of hyperglycemia in fibroblasts in periodontitis tissues. Materials and methods: Fibroblasts were isolated and cultured from normal gums. Gene expression levels were detected by RT-PCR. The protein level was detected by western blotting. Cell viability was determined by MTT assay. To investigate the role of MYDGF, the plasmid was transfected into fibroblasts. The expression levels of cytokines were determined by ELISA. Results: High glucose can down-regulate the expression of MYDGF in human gingival fibroblasts in a time-dependent manner, and decrease the fibroblast activity. SOD level was decreased and MDA level was increased in gingival fibroblasts by high glucose. High glucose up-regulates pro-apoptotic indicator Bax, down-regulates anti-apototic indicator Bcl-2, and increased endoplasmic reticulum stress related indicators Nox 2, GRP78, ATF6, and PERK. In addition, high glucose increased TNF-α, IL-1ß, IL-8 and CXCL1 protein levels in fibroblasts. Our study also found that high glucose inhibits the AKT signaling pathway and activates the nuclear factor κB (NF-κB) pathway. Interestingly, overexpression of MYDGF reversed these effects. Conclusion: MYDGF is down-regulated in gingival fibroblasts induced by high glucose. Overexpression of MYDGF inhibits apoptosis induced by high glucose, inhibits oxidative stress and cytokine secretion of gingival fibroblasts induced by high glucose, and induces AKT pathway activation and NF-κB pathway inhibition.

Isoimperatorin alleviates lipopolysaccharide-induced periodontitis by downregulating ERK1/2 and NF-κB pathways.

Chronic periodontitis is an inflammatory disease characterized by inflammation of the soft tissues of the gums. To combat this disease, more effective drugs are still needed to identify and develop. Isoimperatorin is a kind of a natural compound, which has anti-inflammatory, analgesic, antitumor, antivirus, and other pharmacological effects. However, its possible effects on the progression of chronic periodontitis are still unclear. In this study, we used human periodontal membrane fibroblasts (hPDLCs), human bone marrow-derived macrophages, and found that isoimperatorin reduced hPDLCs viability. In addition, isoimperatorin alleviated the oxidative stress of periodontal membrane cells. Isoimperatorin reduced proinflammatory factor secretion and receptor activator for nuclear factor-κB ligand-induced osteoclast differentiation in periodontal membrane cells. Further, isoimperatorin inhibited the activation of ERK1/2 and nuclear factor-κB pathways. We, therefore, thought isoimperatorin could serve as a promising drug for the treatment of this disease.

First-line contact aspiration versus first-line stent retriever for acute ischemic stroke with M2 occlusion: A systematic review and meta-analysis.

OBJECTIVE: Mechanical thrombectomy is widely used for acute ischemic stroke caused by middle cerebral artery M2 segment occlusion. However, the comparison between contact aspiration (CA) and stent retriever (SR) used as first-line techniques for acute M2 occlusion is still unclear. We aimed to perform a systematic review and meta-analysis on this issue. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement was followed. Three databases (Pubmed, Embase, and Cochrane) were searched. The Newcastle-Ottawa scale was used to assess the risk of bias for the included studies. We focused on two main outcomes, the final successful reperfusion (modified Thrombolysis in Cerebral Infarction mTICI 2b/3) and 90-day functional independence (modified Rankin Scale score 0-2). The meta-analyses were performed using the random-effects models. RESULTS: Seven observational studies were included for systematic review. Only one study indicated a superiority of first-line SR in achieving final successful reperfusion, while the other six studies did not show significant difference between these two techniques. And all the seven studies showed comparable proportion of 90-day functional independence. Five studies were available for meta-analysis with 601 patients (239 received first-line CA, 362 received first-line SR). The pooled results also suggested that the proportion of final successful reperfusion (OR=1.18, 95%CI 0.72-1.93, I2 =0%) and 90-day functional independence (OR=1.18, 95%CI 0.82-1.68, I2 =0%) were comparable between these two strategies. CONCLUSION: For patients with acute M2 occlusion, first-line CA and SR techniques could achieve similar final reperfusion outcomes and 90-day clinical prognosis. Further studies with randomized controlled design are needed.

Atractylenolide-I covalently binds to CYP11B2, selectively inhibits aldosterone synthesis, and improves hyperaldosteronism.

Hyperaldosteronism is a common disease that is closely related to endocrine hypertension and other cardiovascular diseases. Cytochrome P450 11B2 (CYP11B2), an important enzyme in aldosterone (ALD) synthesis, is a promising target for the treatment of hyperaldosteronism. However, selective inhibitors targeting CYP11B2 are still lacking due to the high similarity with CYP11B1. In this study, atractylenolide-I (AT-I) was found to significantly reduce the production of ALD but had no effect on cortisol synthesis, which is catalyzed by CYP11B1. Chemical biology studies revealed that due to the presence of Ala320, AT-I is selectively bound to the catalytic pocket of CYP11B2, and the C8/C9 double bond of AT-I can be epoxidized, which then undergoes nucleophilic addition with the sulfhydryl group of Cys450 in CYP11B2. The covalent binding of AT-I disrupts the interaction between heme and CYP11B2 and inactivates CYP11B2, leading to the suppression of ALD synthesis; AT-I shows a significant therapeutic effect for improving hyperaldosteronism.

First-line contact aspiration versus first-line stent retriever for acute posterior circulation strokes: an updated meta-analysis.

BACKGROUND: Both stent retriever (SR) and contact aspiration (CA) are widely used as first-line strategies for acute posterior circulation strokes (PCS). However, it is still unclear how CA and SR compare as the first-line treatment of acute PCS. Several new studies have been published recently, so we aimed to perform an updated meta-analysis. METHODS: The meta-analysis was conducted according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) statement. Random-effects models were performed to pool the outcomes and the value of I2 was calculated to assess the heterogeneity. RESULTS: Ten observational studies with 1189 patients were included, among whom 492 received first-line CA and 697 received first-line SR. The pooled results revealed that first-line CA could achieve a significantly higher proportion of modified Thrombolysis In Cerebral Infarction (mTICI) 2b/3 (OR 1.90, 95% CI 1.33 to 2.71, I2=0%), mTICI 3 (OR 1.95, 95% CI 1.15 to 3.31, I2=59.6%), first-pass effect (OR 2.91, 95% CI 1.51 to 5.58, I2=0%), lower incidence of new-territory embolic events (OR 0.20, 95% CI 0.05 to 0.83, I2=0%), and shorter procedure time (mean difference -29.4 min, 95% CI -46.8 to -12.0 min, I2=62.8%) compared with first-line SR. At 90-day follow-up, patients subjected to first-line CA showed a higher functional independence (modified Rankin Scale score 0-2; OR 1.38, 95% CI 1.01 to 1.87, I2=23.5%) and a lower mortality (OR 0.71, 95% CI 0.50 to 1.00, p=0.050, I2=0%) than those subjected to first-line SR. CONCLUSIONS: This meta-analysis suggests that the first-line CA strategy could achieve better recanalization and clinical outcomes for acute PCS than first-line SR. Limited by the quality of included studies, this conclusion should be drawn with caution.

Lumican silencing alleviates tumor necrosis factor-α-induced nucleus pulposus cell inflammation and senescence by inhibiting apoptosis signal regulating kinase 1/p38 signaling pathway via inactivating Fas ligand expression.

A recent study has reported that lumican (LUM) is expressed at a high level in the nucleus pulposus specimens from herniated lumbar disc, without description of the specific mechanism. This study was designed to investigate the function and mechanism of LUM in intervertebral disc degeneration (IDD). In this study, human nucleus pulposus cells (hNPCs) cells were challenged with tumor necrosis factor (TNF)-α to establish the IDD in vitro model. After LUM silencing, cell viability was detected using CCK-8 kit, and the expression of inflammatory factors was evaluated using RT-qPCR and ELISA. Flow cytometry and ß-galactosidase staining were used to determine cell cycle and cell senescence. The expression of cycle and senescence-related proteins was evaluated with western blotting. Then, Fas ligand (FasL) was overexpressed and proteins in apoptosis signal regulating kinase 1 (ASK1)/p38 signaling were tested. Finally, GS-4997, an inhibitor of ASK1, was used to explore the regulatory effects of LUM on ASK1/p38 signaling in TNF-α-induced hNPCs. Results indicated that LUM expression was upregulated in TNF-α-challenged hNPCs. LUM gene interference mitigated TNF-α-induced inflammatory response, cell cycle arrest, and senescence of hNPCs. It was then found that LUM silencing could inhibit ASK1/p38 signaling in TNF-α-treated hNPCs, which was reversed by FasL overexpression. Additionally, ASK1/p38 participated in the mediation by LUM of TNF-α-induced inflammation, cell cycle arrest, and senescence of hNPCs. To conclude, interference with LUM effectively mitigated TNF-α-induced inflammatory response, cell cycle arrest, and cell senescence. Further experiments showed the involvement of ASK1/p38 pathway in LUM-mediated NP cell phenotypes through FasL.

Relationship between ABCA1 gene polymorphism and lacunar infarction combined with arteriosclerosis in patients.

Adenosine triphosphate-binding cassette transporter A1 (ABCA1) gene polymorphism in lacunar infarction (LI) combined with arteriosclerosis was investigated. A total of 112 LI patients complicated with arteriosclerosis treated in Ningbo First Hospital from March 2015 to September 2016 were enrolled as observation group. At the same time, 342 healthy subjects were selected from physical examination center to serve as the control group. The ABCA1 gene polymorphism was detected via polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and the susceptibility of ABCA1 gene to LI complicated with atherosclerosis was studied. There were no significant differences in serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp(a)] between the two groups (P>0.05). Levels of triglyceride (TG) and apolipoprotein B (ApoB) in observation group were significantly higher than those in control group, but levels of ApoA-I and high-density lipoprotein cholesterol (HDL-C) were significantly lower in observation group than those in control group (P<0.05). There were no significant differences in the RR, RK and KK frequencies and allele frequency of ABCA1 R219K genotype between the two groups (P>0.05). Moreover, levels of HDL-C increased in the RR, RK and KK genotypes, but were not statistically significant (P>0.05). Levels of TG, TC, LDL-C, ApoA-I, ApoB and Lp(a) showed no significant differences among different genotypes of ABCA1 R219K (P>0.05). Results indicated that ABCA1 R219K polymorphism has no correlation with LI complicated with arteriosclerosis.

The role of basic amino acid surface clusters on the collagenase activity of cathepsin K.

Cathepsin K is a highly potent collagenase in osteoclasts and is responsible for bone degradation. We have previously demonstrated that its unique collagenolytic activity is modulated by glycosaminoglycans that form high molecular weight complexes with the protease. However, mutational analysis of a specific glycosaminoglycan-cathepsin K binding site only led to a 60% reduction of the collagenolytic activity suggesting additional glycosaminoglycan binding sites or other determinants controlling this activity. We identified eight cathepsin K specific arginine/lysine residues that form three positively charged clusters at the bottom part of the protease opposing the active site. These residues are highly conserved among mammalian, avian, and reptilian cathepsin K orthologues and to a lesser degree in amphibian and fish specimens. Mutational analysis of these residues revealed an approximately 50% reduction of the collagenolytic activity when the basic amino acids in cluster 2 (K103, K106, R108, R111) were mutated into alanine residues and resulted in a 100% loss of this activity when the mutations were expanded into cluster 3 (K122, R127). Cluster 1 mutations (K77, R79) had no effect. A partial rescue effect was observed when the hexamutant variant was combined with three mutations in the previously identified glycosaminoglycan binding site (N190, K101, L195K) indicating the relevance of at least two independent interaction sites. Amino acid substitutions in all sites had no effect on the catalytic efficacy of the protease variants as reflected in their unaltered peptidolytic and gelatinolytic activities and their overall protein stabilities. This study suggests that the basic amino acid clusters in cathepsin K are involved in alternative glycoasaminoglycan binding sites, play other roles in the formation of collagenolytically active protease complexes, or contribute in a yet unknown manner to the specific binding to collagen.

Convergence analysis of particle swarm optimizer and its improved algorithm based on velocity differential evolution.

This paper presents an analysis of the relationship of particle velocity and convergence of the particle swarm optimization. Its premature convergence is due to the decrease of particle velocity in search space that leads to a total implosion and ultimately fitness stagnation of the swarm. An improved algorithm which introduces a velocity differential evolution (DE) strategy for the hierarchical particle swarm optimization (H-PSO) is proposed to improve its performance. The DE is employed to regulate the particle velocity rather than the traditional particle position in case that the optimal result has not improved after several iterations. The benchmark functions will be illustrated to demonstrate the effectiveness of the proposed method.

[Effect of transforming growth factor ß(1) and insulin-like growth factor-I on extracelluar matrix synthesis of self-assembled constructs of goat temporomandibular joint disc].

OBJECTIVE: To examine the effects of high and low concentrations of transforming growth factor (TGF) ß(1) and insulin-like growth factor-I (IGF-I) on the extracelluar matrix synthesis of the self-assembled constructs of temporomandibular joint (TMJ) disc. METHODS: The experimental groups of self-assembled constructs were exposed to IGF-I (10, 100 µg/L) and TGF-ß(1) (5, 50 µg/L), the control groups were not added with any growth factors. All groups were examined at 3 and 6 weeks for gross morphological, histological, and biochemical changes. Safranin-O/fast green staining was used to examine glycosaminoglycan (GAG) distribution, picrosirius red and immunohistochemical staining to observe type I collagen distribution. Type I collagen contents were tested by ELISA assay kit, GAG contents were measured by Blyscan GAG assay kit, and the cell numbers were quantified with a Picogreen reagent kit. RESULTS: The growth factor groups all upregulated the matrix synthesis of the self-assembled constructs compared with control groups. TGF-ß(1) (5 µg/L) and IGF-I (10 µg/L) were the two most potent concentration in increasing type I collagen and GAG synthesis and cells proliferation. IGF-I group (10 µg/L) produced nearly 2 times (109.16 ± 5.12 µg) as much type I collagen as the control group (69.13 ± 5.94 µg) at 3 weeks. The matrix contents and the number of the proliferated cells in control group and all GF groups at 6 weeks were more than those at 3 weeks. CONCLUSIONS: IGF-I (10 µg/L) is the most beneficial growth factor and can be applied in tissue-engineering stratigies of the temporomandibular joint disc. At the same time, the exposure time of growth factors is another key factor that affects matrix synthesis of TMJ disc constructs.

[Self-assembly tissue engineering fibrocartilage model of goat temporomandibular joint disc].

OBJECTIVE: To construct self-assembly fibrocartilage model of goat temporomandibular joint disc and observe the biological characteristics of the self-assembled fibrocartilage constructs, further to provide a basis for tissue engineering of the temporomandibular joint disc and other fibrocartilage. METHODS: Cells from temporomandibular joint discs of goats were harvested and cultured. 5.5 x 10(6) cells were seeded in each agarose well with diameter 5 mm x depth 10 mm, daily replace of medium, cultured for 2 weeks. RESULTS: One day after seeding, goat temporomandibular joint disc cells in agarose wells were gathered and began to self-assemble into a disc-shaped base, then gradually turned into a round shape. When cultured for 2 weeks, hematoxylin-eosin staining was conducted and observed that cells were round and wrapped around by the matrix. Positive Safranin-O/fast green staining for glycosaminoglycans was observed throughout the entire constructs, and picro-sirius red staining was examined and distribution of numerous type I collagen was found. Immunohistochemistry staining demonstrated brown yellow particles in cytoplasm and around extracellular matrix, which showed self-assembly construct can produce type I collagen as native temporomandibular joint disc tissue. CONCLUSION: Production of extracellular matrix in self-assembly construct as native temporomandibular joint disc tissue indicates that the use of agarose wells to construct engineered temporomandibular joint disc will be possible and practicable.

Structure-activity analysis of cathepsin K/chondroitin 4-sulfate interactions.

In the presence of oligomeric chondroitin 4-sulfate (C4-S), cathepsin K (catK) forms a specific complex that was shown to be the source of the major collagenolytic activity in bone osteoclasts. C4-S forms multiple contacts with amino acid residues on the backside of the catK molecule that help to facilitate complex formation. As cathepsin L does not exhibit a significant collagenase activity in the presence or in the absence of C4-S, we substituted the C4-S interacting residues in catK with those of cathepsin L. Variants revealed altered collagenolytic activities with the largest inhibitory effect shown by the hexavariant M5. None of the variants showed a reduction in their gelatinolytic and peptidolytic activities when compared with wild-type catK, indicating no structural alteration within their active sites. However, the crystal structure of the M5 variant in the presence of oligomeric C4-S revealed a different binding of chondroitin 4-sulfate. C4-S is not continuously ordered as it is in the wild-type catK·C4-S complex. The orientation and the direction of the hexasaccharide on the catK surface have changed, so that the hexasaccharide is positioned between two symmetry-related molecules. Only one M5 variant molecule of the dimer that is present in the asymmetric unit interacts with C4-S. These substitutions have changed the mode of catK binding to C4-S and, as a result, have likely affected the collagenolytic potential of the variant. The data presented here support our hypothesis that distinct catK/C4-S interactions are necessary for the collagenolytic activity of the enzyme.

Protein kinase C regulates neurite outgrowth in spinal cord neurons.

OBJECTIVE: The functional roles of protein kinase C (PKC) in the neurite outgrowth and nerve regeneration remain controversial. The present study was aimed to investigate the role of PKC in neurite outgrowth, by studying their regulatory effects on neurite elongation in spinal cord neurons in vitro. METHODS: The anterior-horn neurons of spinal cord from embryonic day 14 (E14) Sprague-Dawley (SD) rats were dissociated, purified and cultured in the serum-containing medium. The ratio of membrane-PKC (mPKC) activity to cytoplasm-PKC (cPKC) activity (m/c-PKC) was studied at different time points during culture. RESULTS: Between 3-11 d of culture, the change of m/c-PKC activity ratio and PKC-betaII expression in the neurite were both significantly correlated with neurite outgrowth (r=0.95, P< 0.01; r=0.73, P< 0.01, respectively). Moreover, PMA, an activator of PKC, induced a dramatic elevation in the m/c-PKC activity ratio, accompanied with the increase in neurite length (r=0.99, P< 0.01). In contrast, GF 109203X, an inhibitor of PKC, significantly inhibited neurite elongation, which could not be reversed by PMA. CONCLUSION: PKC activity may be important in regulating neurite outgrowth in spinal cord neurons, and betaII isoform of PKC probably plays a major role in this process.

The crystal and molecular structures of a cathepsin K:chondroitin sulfate complex.

Cathepsin K is the major collagenolytic enzyme produced by bone-resorbing osteoclasts. We showed earlier that the unique triple-helical collagen-degrading activity of cathepsin K depends on the formation of complexes with bone-or cartilage-resident glycosaminoglycans, such as chondroitin 4-sulfate (C4-S). Here, we describe the crystal structure of a 1:n complex of cathepsin K:C4-S inhibited by E64 at a resolution of 1.8 A. The overall structure reveals an unusual "beads-on-a-string"-like organization. Multiple cathepsin K molecules bind specifically to a single cosine curve-shaped strand of C4-S with each cathepsin K molecule interacting with three disaccharide residues of C4-S. One of the more important sets of interactions comes from a single turn of helix close to the N terminus of the proteinase containing a basic amino acid triplet (Arg8-Lys9-Lys10) that forms multiple hydrogen bonds either to the caboxylate or to the 4-sulfate groups of C4-S. Altogether, the binding sites with C4-S are located in the R-domain of cathepsin K and are distant from its active site. This explains why the general proteolytic activity of cathepsin K is not affected by the binding of chondroitin sulfate. Biochemical analyses of cathepsin K and C4-S mixtures support the presence of a 1:n complex in solution; a dissociation constant, K(d), of about 10 nM was determined for the interaction between cathepsin K and C4-S.

Selective inhibition of the collagenase activity of cathepsin K.

Cathepsin K, the main bone degrading protease, and chondroitin 4-sulfate (C4-S) form a complex with enhanced collagenase activity. In this report, we demonstrate the specific inhibition of the collagenase activity of cathepsin K by negatively charged polymers without affecting the overall proteolytic activity of the protease. Three different mechanisms to interfere with cathepsin-catalyzed collagen degradation are discussed: 1) inhibition of the formation of the cathepsin K/C4-S complex, 2) inhibition of the attachment of C4-S to collagen, and 3) masking of the collagenase cleavage sites in collagen. By targeting these interaction sites, collagen degradation can be modulated while the non-collagenolytic activities of cathepsin K remain intact. The main inhibitory effect on collagen degradation is due to the impeding effect on the active cathepsin K/C4-S complex. Essential structural elements in the inhibitor molecules are negative charges which compete with the sulfate groups of C4-S in the cathepsin K/C4-S complex. The inhibitory effect can be controlled by length and charge of the polymers. Longer negatively charged polymers (e.g. polyglutamates, oligonucleotides) tend to inhibit all three mechanisms, whereas shorter ones preferentially affect the cathepsin K/C4-S complex.