Phosphorescence Lifetime Imaging of Labile Zn2+ in Mitochondria via a Phosphorescent Iridium(III) Complex.

Phosphorescence lifetime Zn2+ imaging possesses the advantage over normal fluorescence imaging in offering the more accurate temporal-spatial Zn2+ information. Herein, we report a new phosphorescent cyclometalated Ir(III) complex with a Zn2+-chelator bearing 1,10-phenanthrolin acting as ancillary ligand, Zin-IrDPA, which displays the specific Zn2+-induced enhancement of phosphorescence and phosphorescence lifetime, and the mitochondria-targeting ability. Moreover, its Zn2+-induced phosphorescence lifetime enhancement factor is not affected by medium lipophilicity, viscosity, polarity, and especially ambient oxygen. The reversible tracking of introduced exogenous labile Zn2+ in MCF-7 and HeLa cells via phosphorescence imaging and phosphorescence lifetime imaging (PLIM) have been realized with Zin-IrDPA. Moreover, PLIM with Zin-IrDPA is able to track the SNOC-stimulated endogenous Zn2+ release in mitochondria.

LAMP3 plays an oncogenic role in osteosarcoma cells partially by inhibiting TP53.

BACKGROUND: Osteosarcoma (OS) is a common malignant tumor that predominantly occurs in adolescents. Its most common metastasis is to the lungs. As shown in our earlier study, lysosome-associated membrane glycoprotein 3 (LAMP3) is highly upregulated in metastatic OS. However, its role in the regulation of OS cell viability and apoptosis remains unknown. METHODS: We knocked down and overexpressed LAMP3 in OS cells and assessed the cell viability and apoptosis. Then, we investigated the expression of apoptosis-associated genes to identify the downstream gene(s) of LAMP3. RESULTS: Knockdown of LAMP3 significantly inhibited OS cell viability and promoted apoptosis. TP53, which is involved in the apoptosis pathway, was found to be highly upregulated after knockdown of LAMP3. Overexpression of LAMP3 significantly increased cell viability and abrogated apoptosis. Importantly, subsequent knockdown of TP53 partially suppressed the increased OS cell apoptosis induced by the inhibition of LAMP3, suggesting that TP53 is a key functional downstream gene of LAMP3. CONCLUSIONS: Our findings suggest that LAMP3 promotes OS cell viability and survival by regulating TP53 expression.

Promoter hypermethylation of the cysteine protease RECK may cause metastasis of osteosarcoma.

The present study examined the role of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) promoter hypermethylation as a causative factor in metastasis of osteosarcoma. Using human pathological samples, it is demonstrated that RECK, a cysteine protease that reversibly regulates expression of matrix metalloproteases like matrix metallopeptidase 9 (MMP9), is transcriptionally inhibited in osteosarcoma, especially metastatic variants. This result comes from its promoter hypermethylation, as evaluated in the present study by methylation-specific PCR reaction. The expression of RECK was also significantly diminished in the metastatic variants of osteosarcoma. This downregulation of RECK in advanced grades of osteosarcoma and metastatic grades was also associated with the increased expression of invadosome-specific markers like MMP9, phospho-FAK, and integrins, suggesting the complex contributions of RECK in the prevention of metastasis and its downregulation as a causative factor in osteosarcoma metastasis.

Shikonin protects chondrocytes from interleukin-1beta-induced apoptosis by regulating PI3K/Akt signaling pathway.

Chondrocyte apoptosis is mostly responsible for the development and progression of osteoarthritis. IL-1ß is generally served as an agent that induces chondrocyte apoptosis. Shikonin exerts its anti-inflammatory effect on cartilage protection in vivo. We aimed to explore the protective effect of shikonin on interleukin-1beta (IL-1ß)-induced chondrocyte apoptosis and the potential molecular mechanisms. Chondrocytes were isolated from the joints of newborn Sprague-Dawley rats. The MTT assay and LDH cell death assay were used to determine the cell viability and chondrocyte apoptosis was detected by Annexin-V/PI staining and nucleosomal degradation. The contents of phosphorylated-PI3K (p-PI3k), phosphorylated-Akt (p-Akt), Bcl-2, Bax, and cytochrome c were detected by Western blotting. A quantitative colorimetric assay was used to detect the caspase-3 activity. Our results showed that pretreatment with shikonin (4 µM) inhibited cytotoxicity and apoptosis induced by IL-1ß (10 ng/ml) in chondrocytes. Shikonin pretreatment also decreased the activity of IL-1ß that decreased Bcl-2 expression and levels of p-PI3K and p-Akt, and increased Bax expression, cytochrome c release, and caspase-3 activation. It also reversed the activity of IL-1ß that promoted the synthesis of matrix metalloproteinase-13 and inhibited the expression of tissue inhibitor of metalloproteinase-1 expression, with the net effect of suppressing extracellular matrix degradation. These data suggested that shikonin may protect chondrocytes from apoptosis induced by IL-1ß through the PI3K/Akt signaling pathway, by deactivating caspase-3.

Evaluation of occipitocervical neutral position using lateral radiographs.

BACKGROUND: Intraoperative assessment of neutral occipitocervical balance during a fusion procedure is challenging. We designed this study to introduce a more comprehensive method of evaluating the occipitocervical neutral position using lateral radiographs. METHODS: One hundred neutral lateral cervical spine radiographs interpreted as normal were studied. Cervical spine radiographs were performed using a standard technique. The occipitocervical angle, the occipitocervical distance, and the mandible cervical distance were measured by different observers. RESULTS: A difference analysis was performed between males and females. The mean mandible cervical distances were 11.0 and 11.2 mm in males and females, respectively. The mean occipitocervical distances were 22.0 mm (male) and 19.6 mm (female), and the occipitocervical angles were 47.2° (male) and 45.5° (female). The occipitocervical distance revealed significant differences between males and females (p <0.01). However, there were no significant differences between sexes for the occipitocervical angle or the mandible cervical distance (p >0.01). CONCLUSIONS: This study offers reference values for the occipitocervical angle and occipitocervical distance for the estimation of the occipitocervical neutral position. The introduction of the mandible cervical distance may make the evaluation more direct and more comprehensive during surgery because of its sensitivity to changes in head position.

PPARγ forms a bridge between DNA methylation and histone acetylation at the C/EBPα gene promoter to regulate the balance between osteogenesis and adipogenesis of bone marrow stromal cells.

The balance between osteogenesis and adipogenesis of bone marrow stromal cells is impaired in many human diseases. Knowledge of how to fine-tune this balance is of medical importance. CCAAT/enhancer binding protein α (C/EBPα) has been shown to regulate the balance between osteogenesis and adipogenesis of C3H10T1/2 cells, with epigenetic modifications of the C/EBPα promoter playing an important role. The present study aimed to elucidate the underlying molecular mechanisms. The results showed that peroxisome proliferator-activated receptor γ (PPARγ) binds the -1286 bp/-1065 bp region of the C/EBPα promoter to activate C/EBPα expression during osteogenesis and adipogenesis of C3H10T1/2 cells. DNA hypermethylation in the -1286 bp/-1065 bp region, observed at the terminal stage of osteogenesis, prevented PPARγ binding, and then histone deacetylase 1 (HDAC1) occupied this region to reduce the level of histone acetylation. We regulated the balance between osteogenesis and adipogenesis of mouse bone marrow stromal cells through modulation of DNA methylation and histone acetylation status. In addition, in bone marrow stromal cells from the glucocorticoid-induced osteoporosis (GIO) mouse, hypomethylation of CpG sites, higher binding of PPARγ, acetylated histones 3 and 4, and reduced binding of HDAC1 in the -1286 bp/-1065 bp region of C/EBPα promoter were observed, compared with normal mice. This study provides a deeper insight into the molecular mechanisms underlying the balance between osteogenesis and adipogenesis regulated by C/EBPα in synergy with PPARγ, and suggests a molecular model for how DNA methylation and histone acetylation are linked by PPARγ to regulate differentiation of bone marrow stromal cells.

[Effects of changes in axis length of patella on patellofemoral contact areas and pressures].

OBJECTIVE: To explore the effects of changes in the length of the patella on patellofemoral contact areas and pressures, to provide a theoretical foundation for treatment of lower pole of patella fracture. METHODS: Using homemade-loading equipment, pressure sensitive films of 100 mm x 100 mm in size were placed on the force platform, vertically downward load (0-19.6 N) was given. The pressure-sensitive response curve was obtained by computer image analysis of the pressure-sensitive tablets and calculation. Six male left fresh knee specimens from voluntary donation were placed in homemade-test fixed load device, and the double-layer pressure sensitive film was placed on the patellofemoral joint surface; under loading of 196 N at flexion of 0, 15, 30, 45, 60, 75, 90, 105, 120, and 135 degrees for 2 minutes, respectively, the pressure sensitive film was removed as the control group. Patellas were transected cut and in situ fixed by Kirschner wire and steel-wire as in situ fixation group. Bone fragments obtained from the corresponding 1/6 and 2/6 of contralateral patella, were embedded in the interspace between osteotomy with internal fixation with Kirschner wire and steel-wire respectively as lengthening group. Followed by the amputating patella length of 1/6, 2/6, 3/6 from proximal to distal and internal fixation with Kirschner wire and steel wire by turns as a shortening group. Repeat the above steps of each experiment. By image analysis the pressure sensitive film, the patella joint contact area were measured, and patellar contact pressure (including the peak pressure and average pressure) was calculated according to pressure-sensitive response curve. RESULTS: The actual contact area were significantly smaller in the shortening groups than in the control group at flexion of 30-135 degrees (P < 0.05); the pressure was significantly bigger in shortening 1/6 group at flexion of 0, 15, 60, and 75 degrees, in shortening 2/6 group at flexion of 0 degrees and 75-135 degrees, and in shortening 3/6 group at flexion of 0-30 degrees and 75-135 degrees than in the control group (P < 0.05); the peak pressure was significantly bigger in shortening 1/6 group at flexion of 0, 15, and 60-105 degrees, in shortening 2/6 group at flexion of 0, 15, and 75-105 degrees, and in shortening 3/6 group at flexion of 0, 30, and 60-135 degrees than in the control group (P < 0.05). The actual contact area was significantly smaller in the lengthening groups than in the control group at flexion of 15, 60, and 90 degrees, and it was bigger at flexion of 105, 135 degrees in lengthening 2/6 group than in the control group (P < 0.05); the pressure was significantly bigger in the lengthening groups at flexion of 15-75 degrees than in the control group and it was smaller in the lengthening groups at flexion of 105, 135 degrees, and smaller in lengthening 2/6 group at flexion of 120 degrees (P < 0.05); the peak pressure was significantly smaller in lengthening 1/6 group than in the control group at flexion of 0, 90, and 105 degrees and smaller in lengthening 2/6 group at flexion of 0 degrees (P < 0.05). The actual contact area was significantly bigger in all lengthening groups than in all shortening groups at flexion of 30, 45, and 75-135 degrees (P < 0.05). The pressure was significantly bigger in shortening 1/6 group than in lengthening groups at flexion of 0, 60, and 90 degrees (P < 0.05), in shortening 2/6 group at flexion of 0, 60, and 90-120 degrees (P < 0.05), in shortening 3/6 group at flexion of 0-135 degrees (P < 0.05). The peak pressure was bigger in shortening groups than in lengthening 1/6 group at flexion of 0, 90, and 105 degrees (P < 0.05), bigger than lengthening 2/6 group at flexion of 0 degrees (P < 0.05). CONCLUSION: To treat comminuted fracture of the inferior pole of patella, the partial resection or the late lengthening after preserving the patella has less effect on knee joint function, especially at the patella to be cut less than 1/6 or after surgery lengthening of less than 1/6, while the latter is better than the former. The patella should be preserved as much as possible. If the patellar partial resection is inevitable, the length resection should be less than 1/6, it also can get satisfactory results.

[Current situation of surgical treatment of inferior polar fracture of patella].

OBJECTIVE: To investigate the advance in surgical treatment of inferior pole fracture of patella and to explore the existing problems and further research directions. METHODS: Domestic and foreign literature in recent years on patella fracture was extensively reviewed, the surgical treatment of inferior pole fracture of patella was summarized by combining the research findings with clinical experience. RESULTS: The surgical treatment of inferior pole of patella fractures included retaining the integrity of the patella and partial patellectomy of inferior pole of patella and extending knee installation reconstruction. There were kinds of ways to retain the integrity of the patella, such as circular wire fixation, tension band fixation, NiTi-patella concentrator fixation, basket plate fixation, reforming McLaughlin way and polydioxanone suture net fixation; the latter category is partial patellectomy and extensor device reconstruction. Every surgical way had its advantages and limitations. CONCLUSION: Most studies tend to retain the integrity of the patella, but some researches have shown that partial resection of inferior pole of patella had no significant effect on knee function. It is important to obtain the security excisional range and elongation range postoperative by experiment for regulating the treatment of comminuted fractures of inferior pole of patella.