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Background: Nitrogen-containing bisphosphonate(N-BP)had been found to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells (VSMCs), but the mechanism is not clear. We intend to verify that N-BP induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase(FPPS) to inhibit the osteogenic differentiation and calcification in VSMCs., Methods: β-glycerophosphate (β-GP) was used to induce the osteogenic differentiation and calcification in VSMCs. VSMCs were treated with N-BP or pretreated with downstream products of farnesyl pyrophosphate synthase(FPPS) in mevalonate pathway, such as farnesol (FOH) or geranylgeraniol (GGOH). Alizarin red S staining and determination of calcium content were used to detect calcium deposition.Western Blotting were used to detect expressions of proteins(OPG and RANKL ) and osteogenic marker proteins (Runx2 and OPN)., Results: β-GP induced the osteogenic differentiation and calcification in VSMCs, increased RANKL protein expression and had no significant effect on OPG protein expression. With the treatment of N-BP, the expression of OPG protein was increased and expression of RANKL protein was decreased in VSMCs undergoing osteogenic differentiation and calcification. In addition, N-BP reduced the osteogenic marker proteins (Runx2 and OPN) expression and calcium deposition in VSMCs undergoing osteogenic differentiation and calcification. These effects of N-BP on the osteogenic differentiation and calcification in VSMCs were concentration-dependent, which could be reversed by the downstream products of FPPS, such as FOH or GGOH., Conclusion: N-BP increases OPG expression and decreases RANKL expression via inhibition of FPPS to inhibit the osteogenic differentiation and calcification in VSMCs., (© 2024. The Author(s).)

Background: The role of non-nitrogen-containing bisphosphonates (non-N-BPs) and nitrogen-containing bisphosphonates (N-BPs) in the treatment of atherosclerosis (AS) and vascular calcification (VC) is uncertain. This meta-analysis was conducted to evaluate the efficacy of non-N-BPs and N-BPs in the treatment of AS and VC., Methods: The PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure, and Wanfang databases were searched from their inception to July 5th, 2023. Eligible studies comparing bisphosphonates (BPs) versus no BPs in the treatment of AS and VC were included. The data were analyzed using Review Manager Version 5.3., Results: Seventeen studies were included in this meta-analysis. Twelve were randomized control trials (RCTs), and 5 were nonrandomized studies. Overall, 813 patients were included in the BPs group, and 821 patients were included in the no BPs group. Compared with no BP treatment, non-N-BP or N-BP treatment did not affect serum calcium (P > .05), phosphorus (P > .05) or parathyroid hormone (PTH) levels (P > .05). Regarding the effect on serum lipids, non-N-BPs decreased the serum total cholesterol (TC) level (P < .05) and increased the serum triglyceride (TG) level (P < .01) but did not affect the serum low-density lipoprotein cholesterol (LDL-C) level (P > .05). N-BPs did not affect serum TC (P > .05), TG (P > .05) or LDL-C levels (P > .05). Regarding the effect on AS, non-N-BPs did not have a beneficial effect (P > .05). N-BPs had a beneficial effect on AS, including reducing the intima-media thickness (IMT) (P < .05) and plaque area (P < .01). For the effect on VC, non-N-BPs had a beneficial effect (P < .01), but N-BPs did not have a beneficial effect (P > .05)., Conclusion: Non-N-BPs and N-BPs did not affect serum calcium, phosphorus or PTH levels. Non-N-BPs decreased serum TC levels and increased serum TG levels. N-BPs did not affect serum lipid levels. Non-N-BPs had a beneficial effect on VC, and N-BPs had a beneficial effect on AS., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Introduction: Endoscopic parathyroidectomy(EPTX) has been gradually introduced as a minimally invasive treatment for refractory secondary hyperparathyroidism (SHPT). However, it is uncertain about the efficacy and safety compared between EPTX and open parathyroidectomy (OPTX) for refractory SHPT., Aim: This meta-analysis was conducted to evaluate the efficacy and safety of EPTX and OPTX for secondary hyperp arathyroidism (SHPT)., Material and Methods: Databases including PubMed, EMbase, Cochrane Library, CNKI, and Wanfang were searched. Eligible studies comparing EPTX and OPTX for refractory SHPT were included., Results: Compared with OPTX, EPTX has the shorter hospital stay (p < 0.01) and lower incidences of hoarseness or recurrent laryngeal nerve injury (p = 0.04). There was no significant difference between EPTX and OPTX concerning operation time (p = 0.49), intraoperative blood loss (p = 0.24), postoperative parathyroid hormone levels (p = 0.22), postoperative calcium levels (p = 0.93), postoperative phosphorus levels (p = 0.37), and complications including neck ecchymosis (p = 0.87), subcutaneous haematoma (p = 0.18), and wound infection (p = 0.11)., Conclusions: EPTX and OPTX are both effective methods for refractory SHPT. EPTX had the shorter hospital stay and lower incidences of hoarseness or recurrent laryngeal nerve injury., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2023 Fundacja Videochirurgii.)

Objective: Functional vein end to arterial side (ETS) anastomosis uses vein side to arterial side anastomosis with distal vein ligation, which is different from traditional ETS anastomosis. To date, there are no studies concerning different anastomotic angles of fistula with functional ETS anastomosis. The purpose of the study was to analyze the clinical outcomes concerning different anastomotic angles of functional ETS anastomosis in radiocephalic fistula., Methods: Between January 2018 and December 2020, we performed a prospective cohort study concerning functional ETS anastomosis in radiocephalic fistula. According to vascular anatomy of patients, the anastomosis angles of fistula were designed at 30 ≤ angle ≤ 50°, 50 < angle ≤ 70°, and 135° smooth obtuse angle. The end points were the primary patency rate (PPR), the secondary patency rate (SPR) and the cumulative rate of reintervention (CRR) near anastomotic venous segment., Results: 124 patients with functional ETS anastomosiss were enrolled in this study. Pearson χ 2 test showed that the group of 135°anastomosis angle had the maximum distance between arteries and veins, and the group of 30-50°anastomosis angle had the minimum distance between arteries and veins (P < 0.01). 30-50°anastomosis angle had the highest PPR at 12 months (P = 0.03) and the lowest CRR near anastomotic venous segment at 3 months (P = 0.04) and 12 months (P = 0.01). There were no significant differences among different anastomosis angles concerning the SPR within 12 months (P > 0.05). Kaplan-Meier and log-rank analysis showed that 30-50°anastomosis had the highest PPR (P = 0.03) and the lowest CRR near anastomotic venous segment (P = 0.01). A multivariable Cox model showed anastomotic angle was an independent factor predictive of the PPR (P = 0.04) and the CRR near anastomotic venous segment (P = 0.03). 50-70°anastomosis angle was a risk factor of decreasing PPR (P = 0.03). 50-70° (P = 0.01) and 135° (P = 0.03) anastomosis angle were both obvious risk factors of increasing CRR near anastomotic venous segment., Conclusion: 30-50°were the best anastomotic angles for functional ETS anastomosis, which had the highest PPR and lowest CRR near anastomotic venous segment., (© 2023. The Author(s).)

Objective: Thermal ablation, including microwave ablation (MWA) and radiofrequency ablation (RFA), has been recommended for the treatment of primary hyperparathyroidism (PHPT) and refractory secondary hyperparathyroidism (SHPT). This meta-analysis was conducted to evaluate the efficacy and safety of MWA and RFA in patients with PHPT and refractory SHPT., Methods: Databases including PubMed, EMbase, the Cochrane Library, CNKI (China National Knowledge Infrastructure), and Wanfang were searched from inception to December 5, 2022. Eligible studies comparing MWA and RFA for PHPT and refractory SHPT were included. Data were analyzed using Review Manager software, version 5.3., Results: Five studies were included in the meta-analysis. Two were retrospective cohort studies, and three were RCTs. Overall, 294 patients were included in the MWA group, and 194 patients were included in the RFA group. Compared with RFA for refractory SHPT, MWA had a shorter operation time for a single lesion (P < 0.01) and a higher complete ablation rate for a single lesion ≥ 15 mm (P < 0.01) but did not show a difference in the complete ablation rate for a single lesion < 15 mm (P > 0.05). There were no significant differences between MWA and RFA for refractory SHPT concerning parathyroid hormone (P > 0.05), calcium (P > 0.05), and phosphorus levels (P > 0.05) within 12 months after ablation, except that calcium (P < 0.01) and phosphorus levels (P = 0.02) in the RFA group were lower than those in the MWA group at one month after ablation. There was no significant difference between MWA and RFA concerning the cure rate of PHPT (P > 0.05). There were no significant differences between MWA and RFA for PHPT and refractory SHPT concerning the complications of hoarseness (P > 0.05) and hypocalcaemia (P > 0.05)., Conclusion: MWA had a shorter operation time for single lesions and a higher complete ablation rate for large lesions in patients with refractory SHPT. However, there was no significant difference in efficacy and safety between MWA and RFA in cases of both PHPT and refractory SHPT. Both MWA and RFA are effective treatment methods for PHPT and refractory SHPT., (© 2023. The Author(s).)

Explaining the wetting mechanism of Cu-P brazing materials and Cu remains challenging. This fundamental research aims to reveal the wettability mechanism of Si, Sn, and Zr doping on the interfacial bond strength of the Cu 3 P/Cu system through the first principles study. We carried out several sets of calculations to test the validity of the result; included in the work are those used to establish the interfacial structure and to analyze the effect of doping on the wettability. Specific analysis was carried out in terms of three aspects: the work of adhesion (Wad), the charge density difference, and the density of states (DOS). The calculated results show that doping with Si, Sn, and Zr elements can effectively improve the wettability within the CuP/Cu interface with very high accuracy, and is particularly effective when doped with Zr. These results provide an insightful theoretical guide for enhancing the CuP/Cu system's wettability by adding active elements.

Objective: The objective of this meta-analysis was to compare the efficacy and drug safety of tolvaptan with placebo for autosomal dominant polycystic kidney disease (ADPKD)., Methods: The PubMed, Embase, and Cochrane Library databases were searched from inception to September 10, 2021. Eligible studies comparing tolvaptan and placebo in the treatment of patients with ADPKD were included. Data were analysed using Review Manager Version 5.3., Results: Thirteen studies involving 3575 patients were included in the meta-analysis. Compared with placebo, tolvaptan had a better effect on delaying eGFR decline (MD 1.27, 95% CI 1.24-1.29, P < 0.01) and TKV increase (MD - 3.01, 95% CI - 3.55 to - 2.47, P < 0.01) in ADPKD treatment. Additionally, tolvaptan reduced the incidence of complications such as renal pain (OR 0.71, 95% CI 0.58-0.87, P < 0.01), urinary tract infection (OR 0.69, 95% CI 0.54-0.89, P < 0.01), haematuria (OR 0.68, 95% CI 0.51-0.89, P < 0.01), and hypertension (OR 0.66, 95% CI 0.52-0.82, P < 0.01). However, tolvaptan was associated with a higher incidence rate of adverse events such as thirst (OR 8.48 95% CI 4.53-15.87, P < 0.01), polyuria (OR 4.71, 95% CI 2.17-10.24, P < 0.01), and hepatic injury (OR 4.56, 95% CI 2.51-8.29, P < 0.01)., Conclusion: Tolvaptan can delay eGFR decline and TKV increase and reduce complications such as renal pain, urinary tract infection, haematuria, and hypertension in the treatment of ADPKD. However, tolvaptan increases the adverse effects of thirst, polyuria and hepatic injury., (© 2022. The Author(s).)

Objective: The objective of this meta-analysis was to compare the efficacy and safety of tacrolimus (TAC) monotherapy versus corticosteroid as initial monotherapy in adult-onset minimal change disease (MCD) patients., Methods: Databases including PubMed, Embase, the Cochrane Library, China National Knowledge Infrastructure, and Wanfang database were searched from the inception to March 20, 2021. Eligible studies comparing TAC monotherapy and corticosteroid as initial monotherapy for adult-onset MCD patients were included. Data were analyzed using Review Manager Version 5.3., Results: Four randomized controlled trials (RCTs) involving 196 patients were included in the meta-analysis. For initial monotherapy for adult-onset MCD, TAC and corticosteroid had similar complete remission (OR 1.06, 95% CI 0.47-2.41, P = 0.89), total remission (OR 1.30, 95% CI 0.39-4.35, P = 0.67), relapse rate (OR 0.63, 95% CI 0.28-1.42, P = 0.26). Main drug-related adverse effects of two therapeutic regimens had no difference concerning infection (OR 0.54, 95% CI 0.23-1.27, P = 0.15), glucose intolerance (OR 0.55, 95% CI 0.16-1.84, P = 0.33) and acute renal failure (OR 1.37, 95% CI 0.36-7.31, P = 0.71)., Conclusion: TAC monotherapy is comparable with corticosteroid monotherapy in initial therapy of MCD. To further confirm the conclusion, more large multicenter RCTs are necessary., (© 2022. The Author(s).)

Unconventional machining of WEDM (Wire Electrical Discharge Machining) is playing an increasingly important role in the manufacturing industry. The processing efficiency and resource consumption of this method are research hotspots from the perspective of sustainable development. Energy and CO 2 emissions modeling of process machining have been recognized as an effective and economical ways to achieve energy-saving, emission-reducing and to improve process efficiency. However, the predictive modeling of energy and CO 2 emissions in unconventional machining of WEDM machining has not been thoroughly fully studied. This paper proposes a predictive model of energy consumption and CO 2 emissions in WEDM process considering process characteristics. The application of the energy and CO 2 emissions model proposed in this paper in an example shows that the model's energy consumption prediction accuracy for single part processing reaches 96.5%, and the energy consumption prediction accuracy for batch processing is above 99%. A new standard for cutting fluid substitution with the best machining stability and energy consumption is proposed. In the example, it is also shown that the corners in the geometric structure will reduce the processing energy consumption. The smaller the number of single folding angles, the more energy consumption will be reduced. The processing energy consumption per unit area has a greater deviation when the thickness is low, and the thickness of the workpiece will also affect the life of the electrode wire. It depends on the number of multi-layer stacks and the life of electrode wires; the quality of machine tool auxiliary materials has a greater impact on energy consumption, with a difference of up to 40% in energy consumption. The results of this research can better understand the energy consumption and CO 2 emissions characteristics of the unconventional machining of WEDM., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Resource carbon emission prediction analysis and application for unconventional machining industry: considering the state of the processing process”., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: The objective of this meta-analysis was to compare the efficacy and safety of tacrolimus (TAC) monotherapy versus TAC-corticosteroid combination therapy in idiopathic membranous nephropathy (IMN) patients., Methods: Databases including PubMed, Embase, the Cochrane Library, China National Knowledge Infrastructure, and Wanfang database were searched from inception to January 10, 2021. Eligible studies comparing TAC monotherapy and TAC-corticosteroid combination therapy in IMN patients were included. Data were analysed using Review Manager Version 5.3., Results: Seven studies were included in the meta-analysis. One randomized controlled trial and six cohort studies involving 372 patients were identified. Compared with TAC monotherapy, TAC-corticosteroid had a higher total remission at the sixth month (odd ratio (OR) 0.49, 95% confidence interval (CI) 0.31-0.78, P < .01). The two therapy regimens had similar complete remission rates (OR 0.79, 95% CI 0.43-1.48, P = .47) at the sixth month and similar relapse rates (OR 1.44, 95% CI 0.70-2.92, P = .32). TAC-corticosteroid combination therapy had a higher incidence of infection (OR 0.38, 95% CI 0.18-0.81, P = .01). The two therapy regimens had similar incidences of gastrointestinal symptoms (OR 0.96, 95% CI 0.34-2.70, P = .93), abnormal aminotransferase (OR 0.90, 95% CI 0.34-2.38, P = .84), and glucose intolerance (OR 0.58, 95% CI 0.32-1.07, P = .08)., Conclusion: TAC-corticosteroid combination therapy had a higher total remission rate at the sixth month but had a higher incidence of infection than TAC monotherapy in the treatment of IMN. The two therapeutic regimens had similar relapse rates., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Objective: The objective of this meta-analysis was to compare the efficacy and safety of tacrolimus (TAC) monotherapy versus cyclophosphamide (CTX)-corticosteroid combination therapy in idiopathic membranous nephropathy (IMN) patients., Methods: Databases including the PubMed, Embase, the Cochrane Library, China National Knowledge Infrastructure, and Wanfang databases were searched from inception to October 20, 2020. Eligible studies comparing TAC monotherapy and CTX-corticosteroid combination therapy in IMN patients were included. Data were analyzed using Review Manager Version 5.3., Results: Nine studies were included in the meta-analysis. One randomized controlled trial and eight cohort studies involving 442 patients were identified. Compared with CTX-corticosteroid combination therapy for IMN, TAC monotherapy had higher complete remission (CR) at month 6 (odds ratio [OR] 2.18, 95% confidence interval [CI] 1.35-3.50, P < .01). The 2 therapeutic regimens had similar partial remission (OR 0.69, 95% CI 0.45-1.04, P = .08), total remission (OR 1.38, 95% CI 0.85-2.23, P = 0.19) at month 6, and similar CR (OR 1.64, 95% CI 0.84-3.19, P = .15), partial remission (OR 0.71, 95% CI 0.37-1.38, P = 0.31), and total remission (OR 1.29, 95% CI 0.55-3.01, P = .56) after 1 year. The relapse rate of the TAC group was higher than that of the CTX group, but the difference was not statistically significant (OR 1.85, 95% CI 0.75-4.53, P = .18). There was no difference between the 2 therapeutic regimens concerning glucose intolerance (OR 1.15, 95% CI 0.61-2.14, P = .67), acute renal failure (OR 1.14, 95% CI 0.39-3.33, P = .81), or tremors (OR 4.39, 95% CI 0.75-25.67, P = .10). Incidences of gastrointestinal symptoms (OR 0.29, 95% CI 0.10-0.79, P = .02), infection (OR 0.18, 95% CI 0.08-0.39, P < 0.01), leukopenia (OR 0.14, 95% CI 0.04-0.51, P < .01), and abnormal aminotransferase (OR 0.31, 95% CI 0.13-0.77, P = .01) in the TAC group were all lower than those in the CTX group. Subgroup analysis showed that there was no significant difference between the TAC group and the CTX combined with corticosteroid 0.8 to 1 mg/kg/day group concerning CR at month 6 (P > .05). There was no significant difference between the TAC group and the CTX combined with corticosteroid 0.5 mg/kg/day group concerning abnormal aminotransferase (P > .05)., Conclusion: TAC monotherapy is comparable to CTX-corticosteroid combination therapy for renal remission in IMN patients. TAC monotherapy had a higher CR in the early stage and had fewer drug-related adverse effects. The relapse rate of TAC monotherapy was higher than that of CTX-corticosteroid combination therapy, but the difference was not significant., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The objective of this study was to compare the complications of low-site peritoneal dialysis (PD) catheter placement and traditional open surgery in peritoneal dialysis catheter insertion., Methods: The following databases were searched from inception to September 6, 2019: PubMed, Embase, the Cochrane Library, China National Knowledge Infrastructure, and Wanfang. Eligible studies comparing low-site PD catheter placement and traditional open surgery in peritoneal dialysis catheter insertion were included. The data were analyzed using Review Manager Version 5.3., Results: Seven studies were included in the meta-analysis. A total of 504 patients were included in the low-site PD catheter placement group, and 325 patients were included in the traditional open surgery group. Compared with traditional open surgery, low-site PD catheter placement had a lower incidence rate of catheter displacement (odds ratios [OR] 0.11, 95% CI 0.05-0.22, P < .01) and noncatheter displacement dysfunction (OR 0.11, 95% CI 0.04-0.31, P < .01). However, there was no difference between the 2 catheter insertion methods concerning bleeding (OR 0.53, 95% CI 0.23-1.22, P = .13), PD fluid leakage (OR 0.40, 95% CI 0.15-1.10, P = .07), hypogastralgia (OR 0.95, 95% CI 0.32-2.80, P = .93), peritonitis (OR 0.70, 95% CI 0.32-1.54, P = .38), or exit-site and tunnel infections (OR 0.39, 95% CI 0.14-1.03, P = .06)., Conclusion: Low-site PD catheter placement reduced the risk of catheter displacement and noncatheter displacement dysfunction and did not increase the risk of bleeding, PD fluid leakage, hypogastralgia, peritonitis, or exit site and tunnel infections. Additional large multicenter randomized controlled trials are needed to confirm these conclusions.

Paricalcitol and cinacalcet have been recommended to reduce parathyroid hormone (PTH) levels for patients with secondary hyperparathyroidism (SHPT) and chronic kidney disease (CKD), and they are able to reduce the risk of hypercalcemia and hyperphosphatemia. However, to date, it has remained uncertain which is the better drug. The aim of the present meta-analysis was to evaluate the effects on PTH, calcium and phosphorus metabolism between the two drugs. The PubMed, the Cochrane Library and Embase databases were searched from inception to June 1, 2019 and eligible studies comparing paricalcitol and cinacalcet for SHPT were included. Data were analysed using Review Manager version 5.3. A total of 7 trials from six articles, comprising 456 patients in the paricalcitol group and 412 patients in the cinacalcet group, were included in the meta-analysis. There were no differences in PTH levels [mean difference (MD): 71.82, 95% CI: -185.20-328.85, P=0.58] and phosphorus levels (standard MD: 0.59, 95% CI: -0.82-2.00, P=0.41). The calcium levels in the paricalcitol group were significantly higher than those in the cinacalcet group (MD: 1.10, 95% CI: 0.92-1.28, P<0.05). In conclusion, paricalcitol and cinacalcet exhibited no difference in their efficacy to control of PTH levels, as they were similarly effective in decreasing the PTH levels. They also had comparable efficacy in the management of phosphorus levels. However, cinacalcet produced a significantly greater reduction in serum calcium levels. More large multicentre randomized controlled trials are necessary to confirm the conclusions of the present analysis., (Copyright: © Xu et al.)

Objective: Thermal ablation and parathyroidectomy (PTX) have been recommended for patients with secondary hyperparathyroidism (SHPT). However, it is uncertain which is the better method. The aim of the present meta-analysis was to evaluate the efficacy and surgical complications of the two treatment methods., Methods: The following databases were searched from inception to December 31, 2018: PubMed, EMBASE, the Cochrane Library, CNKI, and Wanfang. Eligible studies comparing thermal ablation and PTX for SHPT were included. Data were analysed using Review Manager Version 5.3., Results: Six studies were included in the meta-analysis. Four cohort studies and two randomized controlled trials involving 326 patients with SHPT were identified. There was no difference concerning parathyroid hormone (PTH) levels (MD 58.04, 95% CI -17.60-133.68, P = 0.13), calcium levels (MD -0.07, 95% CI -0.17-0.04, P = 0.21), phosphorus levels (MD 0.21, 95% CI -0.18-0.61, P = 0.29), or hoarseness (OR 0.53, 95% CI 0.24-1.16, P = 0.11) between the two surgical methods. Compared with PTX, thermal ablation reduced the risk of hypocalcaemia (OR 0.23, 95% CI 0.11-0.47, P < 0.01). However, thermal ablation increased the risk of SHPT persistence and/or recurrence compared with PTX (OR 4.24, 95% CI 1.44-15.76, P = 0.03)., Conclusion: Thermal ablation and PTX were effective surgical approaches for SHTP. Thermal ablation reduced the risk of hypocalcaemia and increased the risk of SHPT persistence and recurrence. More large multicentre randomized controlled trials are necessary to confirm the conclusions., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxic side product formed in the chemical synthesis of desmethylprodine opioid analgesic, which induces Parkinson disease. Monoamine oxidase B, present in the mitochondrial outer membrane of glial cells, catalyzes the oxidation of MPTP to the toxic 1-methyl-4-phenylpyridinium ion (MPP(+)), which then targets the dopaminergic neurons causing neuronal death. Here, we demonstrate that mitochondrion-targeted human cytochrome P450 2D6 (CYP2D6), supported by mitochondrial adrenodoxin and adrenodoxin reductase, can efficiently catalyze the metabolism of MPTP to MPP(+), as shown with purified enzymes and also in cells expressing mitochondrial CYP2D6. Neuro-2A cells stably expressing predominantly mitochondrion-targeted CYP2D6 were more sensitive to MPTP-mediated mitochondrial respiratory dysfunction and complex I inhibition than cells expressing predominantly endoplasmic reticulum-targeted CYP2D6. Mitochondrial CYP2D6 expressing Neuro-2A cells produced higher levels of reactive oxygen species and showed abnormal mitochondrial structures. MPTP treatment also induced mitochondrial translocation of an autophagic marker, Parkin, and a mitochondrial fission marker, Drp1, in differentiated neurons expressing mitochondrial CYP2D6. MPTP-mediated toxicity in primary dopaminergic neurons was attenuated by CYP2D6 inhibitor, quinidine, and also partly by monoamine oxidase B inhibitors deprenyl and pargyline. These studies show for the first time that dopaminergic neurons expressing mitochondrial CYP2D6 are fully capable of activating the pro-neurotoxin MPTP and inducing neuronal damage, which is effectively prevented by the CYP2D6 inhibitor quinidine.

A number of studies show that mitochondrial DNA (mtDNA) depletion and attendant activation of retrograde signaling induces tumor progression. We have reported previously that activation of a novel nuclear factor-Kappa B pathway is critical for the propagation of mitochondrial retrograde signaling, which induces both phenotypic and morphological changes in C2C12 myoblasts and A549 lung carcinoma cells. In this study, we investigated the role of stress-induced nuclear factor-Kappa B in tumor progression in xenotransplanted mice. We used a retroviral system for the inducible expression of small interfering RNA against IkBα and IkBβ mRNAs. Expression of small interfering RNA against IkBβ markedly impaired tumor growth and invasive ability of mtDNA-depleted C2C12 myoblasts and also thwarted anchorage-independent growth of the cells. Knockdown of IkBα mRNA, however, did not have any modulatory effect in this cell system. Moreover, expression of small interfering RNA against IkBβ reduced the expression of marker genes for retrograde signaling and tumor growth in xenografts of mtDNA-depleted cells. Our findings demonstrate that IkBβ is a master regulator of mitochondrial retrograde signaling pathway and that the retrograde signaling plays a role in tumor growth in vivo. In this regard, IkBβ supports the tumorigenic potential of mtDNA-depleted C2C12 cells.

Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (psim) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca2+ [Ca2+]c as part of this signaling cascade activates Ca2+ responsive phosphatase, calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis is a phenotypic hallmark of C2C12 skeletal muscle cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFkappaB (cRel:p50) pathway, NFAT, CREB and C/EBPdelta. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFkappaB (cRel:p50), C/EBPdelta, CREB, and NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional coactivator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes., (Copyright © 2010. Published by Elsevier B.V.)

Constitutively expressed human cytochrome P450 2D6 (CYP2D6; EC 1.14.14.1) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. In the present study, we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23-33 and that the positively-charged residues at positions 24, 25, 26, 28 and 32 are required for mitochondrial targeting. The importance of the positively-charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1'-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall, these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism.

The NFkappaBs regulate an array of physiological and pathological processes, including propagation of mitochondrial respiratory stress signaling in mammalian cells. We showed previously that mitochondrial stress activates NFkappaB using a novel calcineurin-requiring pathway that is different from canonical or non-canonical pathways. This study shows that IkappaBbeta is essential for the propagation of mitochondrial stress signaling. Knock down of IkappaBbeta, but not IkappaBalpha, mRNA reduced the mitochondrial stress-mediated activation and nuclear translocation of cRel:p50, inhibiting expression of nuclear target genes RyR1 and cathepsin L. IkappaBbeta mRNA knock down also reduced resistance to staurosporine-induced apoptosis and decreased in vitro invasiveness. Induced receptor switching to insulin-like growth factor-1 receptor and increased glucose uptake are hallmarks of mitochondrial stress. IkappaBbeta mRNA knock down selectively abrogated the receptor switch and altered tubulin cytoskeletal organization. These results show that mitochondrial stress signaling uses an IkappaBbeta-initiated NFkappaB pathway that is distinct from the other known NFkappaB pathways. Furthermore, our results demonstrate the distinctive physiological roles of the two inhibitory proteins IkappaBbeta and IkappaBalpha.

Prolactin receptors (PRLr) expressed in a majority of breast cancer are activated by prolactin and growth hormone. The PRLr is commonly stabilized in human breast cancer due to decreased phosphorylation of residue Ser(349), which, when phosphorylated, recruits the beta Trcp E3 ubiquitin ligase and facilitates PRLr degradation. Here, we show that constitutive oncogenic signaling downstream of ErbB2 and Ras stabilizes PRLr via inhibitory phosphorylation of glycogen synthase kinase-3beta (GSK3 beta) on Ser(9). Importantly, inactivation of GSK3 beta correlates with elevated levels of PRLr protein in clinical human breast cancer specimens. Additional studies using pharmacologic, biochemical, and genetic approaches reveal that GSK3 beta is a bona fide PRLr kinase that phosphorylates PRLr on Ser(349) and is required for the recognition of PRLr by beta Trcp, as well as for PRLr ubiquitination and degradation.

beta-Transducin repeat-containing proteins (beta-TrCP) serve as substrate recognition component of E3 ubiquitin ligases that control stability of important regulators of cell cycle and signal transduction. beta-TrCP function is essential for the induction of nuclear factor kappaB transcriptional activities, which play a key role in proliferation and survival of cancer cells and are often constitutively up-regulated in human breast cancers. Here we show that inhibition of beta-TrCP either by RNAi approach or by forced expression of a dominant-negative beta-TrCP mutant suppresses growth and survival of human breast cancer cells. In addition, inhibition of beta-TrCP augments the antiproliferative effects of anticancer drugs such as doxorubicin, tamoxifen, and paclitaxel on human mammary tumor cells. These data provide the proof of principle that targeting beta-TrCP might be beneficial for anticancer therapies.

Ubiquitin-dependent degradation of hormone receptors is emerging as a key mechanism that regulates the magnitude and duration of hormonal effects on cells and tissues. The pituitary hormone prolactin (PRL) is involved in regulating cell differentiation, proliferation, and survival. PRL engages its receptor (PRLR) to initiate various signaling cascades, including the phosphorylation and activation of Stat5. We found that PRL promotes interaction between PRLR and the F-box protein beta-TrCP2, which functions as a substrate recognition subunit of the SCF(beta-TrCP) E3 ubiquitin ligase. This interaction requires PRLR phosphorylation and the integrity of serine 349 within a conserved motif, which is similar to conserved motifs present in other substrates of SCF(beta-TrCP). The PRLR(S349A) mutant is resistant to ubiquitination and is more stable than its wild-type counterpart. Phosphorylated PRLR undergoes ubiquitination by SCF(beta-TrCP) in vitro. Knockdown of beta-TrCP expression inhibits the ubiquitination and degradation of PRLR and promotes PRL-dependent phosphorylation of Stat5 as well as Stat5-dependent transcription in cells. Furthermore, the activation of Stat5 and the stimulation of cell growth by PRL are augmented in cells expressing the PRLR(S349A) mutant. These data indicate that PRLR is a novel SCF(beta-TrCP) substrate and implicate beta-TrCP as an important negative regulator of PRL signaling and cellular responses to this hormone.

The Epstein-Barr virus latent membrane protein 1 (LMP1) is pivotal in the transforming activity of this virus. We found that the common LMP1-95-8 variant interacts with Homologue of Slimb (HOS), a receptor for the SCFHOS/betaTrCP ubiquitin-protein isopeptide ligase (E3) via one canonical and one cryptic HOS recognition site. These sites are mutated or deleted in the tumor-derived LMP1-Cao variant, which did not bind to HOS. Mutations within these sites on LMP1-95-8 abrogated HOS binding and increased transforming activity of LMP1. HOS did not regulate stability of LMP1-95-8 unless it was mutated to bear additional lysine residues near the cryptic motif. LMP1 proteins that could not bind to HOS exhibited an increased ability to induce IkappaB degradation and NF-kappaB-mediated transcription without further increase in activation of IkappaB kinases. Expression of LMP1-95-8 reduced the levels of endogenous HOS available to interact with phosphorylated IkappaBalpha. Degradation of IkappaBalpha and dose dependence of NF-kappaB activation by LMP1-95-8 were promoted by co-expression of HOS. Our data suggest that LMP1-95-8 is a pseudo-substrate of SCFHOS/betaTrCP E3 ubiquitin ligase and that interaction between LMP1 and HOS restricts the extent of LMP1-induced NF-kappaB signaling. We discuss the potential role of this mechanism in transforming and cytostatic effects of LMP1 variants in cells and Epstein-Barr virus-associated tumors.

Down-regulation of activated signaling receptors in response to their ligands plays a key role in restricting the extent and duration of the signaling. Mechanisms underlying down-regulation of the type I interferon receptor consisting of IFNAR1 and IFNAR2 subunits remain largely unknown. Here we show that IFNAR1 interacts with the Homolog of Slimb (HOS) F-box protein in a phosphorylation-dependent manner, and that this interaction is promoted by interferon alpha (IFNalpha). IFNAR1 is ubiquitinated by the Skp1-Cullin1-HOS-Roc1 (SCF(HOS)) ubiquitin ligase in vitro. HOS expression and activities are required for IFNalpha-stimulated ubiquitination of IFNAR1, endocytosis of the type I interferon receptor, down-regulation of IFNAR1 levels, and IFNAR1 proteolysis via the lysosomal pathway. Furthermore, modulations of HOS activities affect the extent of Stat1 phosphorylation and Stat-mediated transcriptional activities as well as the extent of antiproliferative effects of type I interferons. These findings characterize SCF(HOS) as an E3 ubiquitin ligase that is essential for ubiquitination, proteolysis and down-regulation of IFNAR1, and implicate HOS in the regulation of cellular responses to IFNalpha.

Homologue of Slimb (HOS) is the substrate-recognizing component of the SCF(HOS)-Roc1 E3 ubiquitin protein ligase. This ligase mediates ubiquitination of the inhibitor of NF-kappaB transcription factor (IkappaB). We have found that HOS is highly expressed in a number of human cancer cell lines. The rates of the HOS gene transcription as well as HOS mRNA and protein levels were up-regulated in cells treated with mitogens or transfected with the inducers of mitogen-activated protein kinase pathway. Conversely, mitogen withdrawal strikingly reduced HOS levels during differentiation of mouse myoblasts. Activators of mitogen-activated protein kinase accelerated IkappaBalpha degradation and increased NF-kappaB transcriptional activity. Inhibition of HOS function via expression of dominant negative HOS (HOS(DeltaF)) initiated mouse myoblast differentiation and prevented Ras-mediated acceleration of IkappaBalpha degradation as well as NF-kappaB trans-activation and transformation of NIH3T3 cells. These data link the induction of HOS in proliferating cells with mitogen-signaling-dependent inhibition of cell differentiation and promotion of cell transformation.

BetaTrCP and HOS are closely related F-box proteins, which play key roles in ubiquitination and degradation of beta-catenin and IkappaB through associating with those phosphorylated substrates and recruiting SCF E3 ubiquitin ligase. Here we report that activation of Wnt/beta-catenin signal transduction pathway elevates betaTrCP levels but inhibits expression of HOS in 293T cells. Similar disparity is likely to exist in human colorectal tumors. In the NIH3T3 cells, which express HOS, but not betaTrCP, Wnt/beta-catenin signaling leads to inhibition of HOS promoter activity and NF-kappaB-driven transcription as well as to stabilization of beta-catenin. These results indicate that expression and activities of HOS are negatively regulated by Wnt/beta-catenin pathway.