Corresponding Changes of Autophagy-Related Genes and Proteins in Different Stages of Knee Osteoarthritis: An Animal Model Study.

OBJECTIVE: To investigate the effect of autophagy expression levels of different weight-bearing states and different stages of osteoarthritis in animal models, as well as the corresponding mechanisms. METHODS: We used the male Sprague-Dawley (SD) rats (12-week-old, SPF) to establish the OA animal models by modified Hulth method, and grouped animal models according to the length of time after surgery and different weight-bearing areas. RT-qPCR was carried out for detection of autophagy-related genes such as Atg7, Atg12, P62, etc. Western blot analysis was used to detect the expression levels of corresponding autophagy-related proteins such as LC3B, P62, etc. T test was performed for statistical analysis to compare different groups, while the differences were deemed statistically significant with P < 0.05. Transmission electron microscopy was used to observe the autophagosome to demonstrate the level of autophagy expression and the status of the chondrocytes. RESULTS: The results of the RT-qPCR testing showed that when the weight-bearing cartilage of the 4-week group (relatively mild) was compared with that of the 10-week group (relatively severe), there were statistically significant differences in all the genes tested, in detail: Atg3 (P < 0.01), Atg7 (P < 0.01), Atg12 (P < 0.01), P62 (P < 0.0001). The expression of autophagy-related mRNA in the 4-week group is increased compared with that of the 10-week group. As for the expression of proteins, Western blotting showed that in the comparison between the 4- and the 10-week groups, statistically significant results include Atg12 (P < 0.01) in the non-weight-bearing area, with decreased autophagy in the 10-week group compared with that of the 4-week group, while expression of LC3B (P < 0.05) protein was significantly higher in the 4-week group than in the control in the non-weight-bearing area. The expression of LC3B (P < 0.0001) and P62 (P < 0.05) in the 10-week group were higher than that of the control. Transmission electron microscope showed that autophagy in the weight-bearing area is stronger than that in the non-weight-bearing area, and autophagy in the 4-week group is stronger than in the 10-week group for the weight-bearing area. CONCLUSIONS: The expression of autophagy varies during different stages of osteoarthritis, in which the autophagy is stronger in the early stage of osteoarthritis, and gradually decreases with the progression of the disease. Autophagy in different weight-bearing areas may also be different.

Theoretical Insights into the Cotransport Mechanism of GSH with Anticancer Drugs by MRP1.

The multidrug resistance protein MRP1 is an ATP binding cassette (ABC) transporter that confers resistance to many anticancer drugs and regulates redox homeostasis, inflammation, and hormone secretion. MRP1 actively transports compounds across cell membranes, and the presence of glutathione (GSH) is required in many cases. However, the process of MRP1-mediated substrate transportation has been poorly understood. With extensive molecular dynamics simulations, we have found a sandwich-like structure which is generated by GSH, a transmembrane α-helices 11 (TM11)-TM17 axis, and anticancer drugs. This structure is crucial in MRP1 transportation. It triggers the motion of TM11 and TM17, followed by the movement of nucleotide-binding domains 1 (NBD1) and 2 (NBD2), and finally an occluded structure is formed. Trp1246, Lys332, and Phe594 were identified as the main contributors in the formation of the sandwich-like structure. Our findings clearly explain the synergy of GSH with an anticancer drug in MRP1 transportation and have significant meanings for the rational design of novel inhibitors against MRP1.

Effects of Tumor Necrosis Factor Alpha on the Expression of Programmed Cell Death Factor 5 in Arthritis.

OBJECTIVE: To investigate the effect of tumor necrosis factor alpha (TNF-α) on the proliferation of fibroblast-like synoviocytes (FLS) and the expression of programmed cell death factor 5 (PDCD5) in an inflammatory microenvironment, for the further understanding of the mechanism of action of TNF-α in promoting the proliferation of synovial cells and the apoptosis of the chondrocytes. METHODS: Articular carriage specimens were obtained from 21 cases with osteoarthritis and 12 cases with femoral neck fractures as healthy controls during arthroplasties. The expression of PDCD5 was evaluated by immunofluorescence analyzed by mean option density (MOD) detected using the software ImagePro Plus. Real-time PCR was performed to evaluate the transcriptions of PDCD5 and TNF-α in synovium. FLS cells derived from rheumatoid arthritis patients were cultured in vitro and incubated with different concentrations of TNF-α. The effects of TNF-α at different concentrations on the proliferation of FLS cells were detected by Cell Counting Kit-8 (CCK-8) assay to evaluate the cell proliferation rate. After incubation with the absence or presence of recombinant human TNF-α at different concentrations, the FLS cells were isolated for detection of PDCD5 protein and PDCD5 gene. The expression of PDCD5 protein was detected by western-blot and the transcription of PDCD5 gene from the cells was detected by real-time quantitative PCR. RESULTS: The MOD of PDCD5 as well as TNF-α of osteoarthritis cartilage sections were significantly increased compared with those of the controls, and in synovium there was a positive correlation between transcriptions of their mRNA. When the concentration of TNF-α was 1 ng/mL, the cell proliferation rate was not significantly different from that of the control group (P = 0.592), while the proliferation of FLS cells was significantly promoted when the concentration of TNF-α was 5, 10, 15, or 20 ng/mL, and the proliferation-promoting rates were 35.64% ± 6.96%, 48.72% ± 7.69%, 45.60% ± 8.85%, and 39.32% ± 6.18%, respectively (P < 0.01). The transcription of PDCD5 gene was significantly downregulated, which was 80.44% ± 4.07% and 84.30% ± 5.48%, respectively (P < 0.05), in the FLS cells incubated with TNF-α at the concentration of 10 and 15 ng/mL for 24 h. When the concentration of TNF-α was 1, 5, or 20 ng/mL, the transcription of PDCD5 mRNA in FLS cells was not significantly different from that in the control group (P > 0.05). The expression of PDCD5 protein was only significantly downregulated when the concentration of TNF-α was 10 ng/mL (P < 0.01), while the expression of PDCD5 protein in FLS cells was not significantly different from that in the control group (P > 0.05). CONCLUSION: The expression of PDCD5 as well as TNF-α in osteoarthritis cartilage and synovium was significantly higher than in healthy tissues, and TNF-α can promote the proliferation of FLS cells in patients with rheumatoid arthritis, and inhibit the expression of PDCD5. PDCD5 may be involved in the abnormal proliferation of synoviocytes and the degeneration of chondrocytes stimulated by TNF-α.

Fragment-centric topographic mapping method guides the understanding of ABCG2-inhibitor interactions.

Understanding protein-ligand interactions is crucial to drug discovery and design. However, it would be extremely difficult for the proteins which only have one available apo structure but multiple binding sites. To address this constraint, a fragment-centric topographic mapping method (AlphaSpace software) was employed to map out concave interaction pockets at the assigned protein region. These pockets are used as complementary spaces to screen the known inhibitors for this specific binding site and to guide the molecular docking pose selection as well as protein-ligand interaction analysis. By mapping the shape of central cavity surface, we have tested the strategy against a multi-drug resistant transmembrane protein-ABCG2 to assist in generating a pharmacophore model for its inhibitors that is based on the structure of apo. Classical molecular simulation and accelerated molecular simulation are used to verify the accuracy of inhibitor screening and binding pose selection. Our study not only has gained insight for the development of novel specific ABCG2 inhibitors, but also has provided a general strategy in describing protein-ligand interactions.

Nitrate Accumulation and Leaching in Surface and Ground Water Based on Simulated Rainfall Experiments.

To evaluate the process of nitrate accumulation and leaching in surface and ground water, we conducted simulated rainfall experiments. The experiments were performed in areas of 5.3 m2 with bare slopes of 3° that were treated with two nitrogen fertilizer inputs, high (22.5 g/m2 NH4NO3) and control (no fertilizer), and subjected to 2 hours of rainfall, with. From the 1st to the 7th experiments, the same content of fertilizer mixed with soil was uniformly applied to the soil surface at 10 minutes before rainfall, and no fertilizer was applied for the 8th through 12th experiments. Initially, the time-series nitrate concentration in the surface flow quickly increased, and then it rapidly decreased and gradually stabilized at a low level during the fertilizer experiments. The nitrogen loss in the surface flow primarily occurred during the first 18.6 minutes of rainfall. For the continuous fertilizer experiments, the mean nitrate concentrations in the groundwater flow remained at less than 10 mg/L before the 5th experiment, and after the 7th experiment, these nitrate concentrations were greater than 10 mg/L throughout the process. The time-series process of the changing concentration in the groundwater flow exhibited the same parabolic trend for each fertilizer experiment. However, the time at which the nitrate concentration began to change lagged behind the start time of groundwater flow by approximately 0.94 hours on average. The experiments were also performed with no fertilizer. In these experiments, the mean nitrate concentration of groundwater initially increased continuously, and then, the process exhibited the same parabolic trend as the results of the fertilization experiments. The nitrate concentration decreased in the subsequent experiments. Eight days after the 12 rainfall experiments, 50.53% of the total nitrate applied remained in the experimental soil. Nitrate residues mainly existed at the surface and in the bottom soil layers, which represents a potentially more dangerous pollution scenario for surface and ground water. The surface and subsurface flow would enter into and contaminate water bodies, thus threatening the water environment.

Modeling the impact of soil and water conservation on surface and ground water based on the SCS and Visual MODFLOW.

Soil and water conservation measures can impact hydrological cycle, but quantitative analysis of this impact is still difficult in a watershed scale. To assess the effect quantitatively, a three-dimensional finite-difference groundwater flow model (MODFLOW) with a surface runoff model-the Soil Conservation Service (SCS) were calibrated and applied based on the artificial rainfall experiments. Then, three soil and water conservation scenarios were simulated on the sand-box model to assess the effect of bare slope changing to grass land and straw mulching on water volume, hydraulic head, runoff process of groundwater and surface water. Under the 120 mm rainfall, 60 mm/h rainfall intensity, 5 m(2) area, 3° slope conditions, the comparative results indicated that the trend was decrease in surface runoff and increase in subsurface runoff coincided with the land-use converted from bare slope to grass land and straw mulching. The simulated mean surface runoff modulus was 3.64×10(-2) m(3)/m(2)/h in the bare slope scenario, while the observed values were 1.54×10(-2) m(3)/m(2)/h and 0.12×10(-2) m(3)/m(2)/h in the lawn and straw mulching scenarios respectively. Compared to the bare slope, the benefits of surface water reduction were 57.8% and 92.4% correspondingly. At the end of simulation period (T = 396 min), the simulated mean groundwater runoff modulus was 2.82×10(-2) m(3)/m(2)/h in the bare slope scenario, while the observed volumes were 3.46×10(-2) m(3)/m(2)/h and 4.91×10(-2) m(3)/m(2)/h in the lawn and straw mulching scenarios respectively. So the benefits of groundwater increase were 22.7% and 60.4% correspondingly. It was concluded that the soil and water conservation played an important role in weakening the surface runoff and strengthening the underground runoff. Meanwhile the quantitative analysis using a modeling approach could provide a thought for the study in a watershed scale to help decision-makers manage water resources.

Programmed cell death 5 correlates with disease activity and interleukin-17 in serum and synovial fluid of rheumatoid arthritis patients.

BACKGROUND: Programmed cell death 5 (PDCD5) is a novel apoptotic regulatory gene that promotes apoptosis in various tumor cells. Studies have shown that PDCD5 accelerates the apoptosis of synoviocytes in vitro, implying a potential role in rheumatoid arthritis (RA) pathogenesis. This study examined the expression of PDCD5 in serum and synovial fluid of RA patients, its effect on the expression of inflammatory cytokine, interleukin-17 (IL-17), and the assessment of disease activity in RA. METHODS: PDCD5 and IL-17 levels in serum and synovial fluid from 18 patients with RA and 22 patients with osteoarthritis (OA) were detected using enzyme-linked immunosorbent assay (ELISA). Concentrations of serum PDCD5 in 40 healthy people were also detected as controls. As disease activity indices, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and X-ray grading scale were also evaluated. RESULTS: Serum and synovial fluid PDCD5 levels in RA patients were significantly higher than those in OA and healthy controls. Serum PDCD5 level was inversely correlated to CRP and ESR, and was significantly higher in the RF negative group than in the positive group. PDCD5 level was also negatively correlated with IL-17 levels both in serum and synovial fluid of RA patients. However, differences in synovial fluid PDCD5 level from RA patients at different Larsen stages were not detectable. CONCLUSIONS: PDCD5 affects RA pathogenesis. Insufficient apoptosis of fibroblast-like synoviocytes and inflammatory cells in RA could increase the expression of PDCD5 protein. As PDCD5 levels correlated negatively with disease activity indices and IL-17 level, PDCD5 could become a target in the diagnosis and treatment of RA.

[Autogeneous bone graft in the treatment of total knee arthroplasty for severe genu varus with tibial plateau bone defect].

OBJECTIVE: To introduce the method and experience of autogeneous bone graft for tibial plateau reconstruction during the procedure of total knee arthroplasty for severe varus knees with bone defect in the medial tibial plateau. METHODS: From April 2007 to March 2011, 19 knees of 16 osteoarthritic patients who had serious genu varus with bone defect in medial tibial plateau underwent primary total knee arthroplasty, their mean varus degree being 32° (25°to 45°), and average age 66±8 years (52 to 77 years). Their preoperative knee functions are as follows: the average range of motion (ROM) was 62°(37° to 90°); average knee society scure(KSS) knee score was 18 points (-24 to 41 points); average function score was 13 points (-21 to 43 points). During operation, the slope bone defect of the medial tibial plateau was dressed into step-shape horizontal bone defect by osteotomy, and then the defect was restored with the resected tibial plateau autograft whose thickness and shape were matched in advance; and the high-intensity cortical part of the autograft was placed on the rim, to sustain the tibial prosthesis; and a lateral pressure from the rim had to be maintained to the autograft until the cement under tibial prosthesis solidified. The long-stem tibial prostheses were used in 3 patients ( 3 knees ). All knee prostheses were fixed using antibiotic bone cements. RESULTS: The average follow-up after TKR was 25 months (3 to 50 months), the average ROM was 112° (95° to 125°); average KSS score 86 points (71 to 93 points), and knee function score 88 points ( 74 to 96 points). The nonunion, shift, fracture of the autologous graft bone were not found; no tibial prosthesis became loose, either; no knee was revised for delayed infection or recurrent varus due to autologous graft bone absorption. CONCLUSION: In TKA for severe varus osteoarthritic patients with bone defect in the tibial plateau, there are various ways to regain its stability, and reconstruction is adopted by using step-shape allograft in this paper. This method can not only restore the integrity of the tibial plateau, providing good initial stability of the prostheses, and exempting from internal fixations, but also offer more reliable compatibility than other methods, reducing postoperative infection rates, and obtaining satisfied initial curative effect.

Some insights into mechanism for binding and drug resistance of wild type and I50V V82A and I84V mutations in HIV-1 protease with GRL-98065 inhibitor from molecular dynamic simulations.

The single mutations I50V, V82A and I84V are considered as the key residue mutations of the HIV-1 protease drug resistance. The rank of calculated absolute binding free energies using MM-PBSA method is in excellent agreement with experimental result. Enthalpic and entropic balance is analyzed to explain resistance in I50V and V82A having a higher entropic contribution than in the wild type (WT) complex. The reduced van der Waals energy explains the drug resistance of I84V to GRL-98065. Detailed binding free energies between GRL-98065 and individual protein residues are calculated to provide insights into the inhibitor-protein binding and drug-resistant mechanism. Our results show I50V and V82A have larger structural changes than I84V compared with WT.