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1.  Low Wear of a Second-generation Highly Crosslinked Polyethylene Liner: A 5-year Radiostereometric Analysis Study 
Background
A sequentially irradiated and annealed, second-generation highly crosslinked polyethylene (XLPE) liner was introduced clinically in 2005 to reduce in vivo oxidation. This liner design has also been shown to reduce wear in vitro when compared with conventional and first-generation crosslinked liners. To date, there is only one study reporting an in vivo wear rate of this liner at 5 years’ followup. However, that study used measurements made from plain radiographs, which have limited sensitivity, particularly when monitoring very low amounts of wear.
Questions/purposes
What is the amount and direction of wear at 5 years using radiostereometric analysis (RSA) in patients who had THAs that included second-generation XLPE?
Methods
We prospectively reviewed 21 patients who underwent primary cementless THA with the same design of XLPE acetabular liner and 32-mm articulation. Tantalum markers were inserted during surgery and all patients had RSA radiographs at 1 week, 6 months, and 1, 2, and 5 years postoperatively. Femoral head penetration within the acetabular component was measured with UmRSA® software. One patient died and two had incomplete radiographs leaving 18 radiographic series for analysis.
Results
The mean amounts of proximal, two-dimensional, and three-dimensional head penetration between 1 week and 5 years were 0.018, 0.071, and 0.149 mm, respectively. The mean proximal, two-dimensional, and three-dimensional wear rates calculated between 1 year and 5 years were all less than 0.001 mm/year with no patient recording a wear rate of more than 0.040 mm/year.
Conclusions
The head penetration of a second-generation XLPE liner remained low at 5 years and the wear rate calculated after the first year was low in all directions. This low level of wear remains encouraging for the future clinical performance of this material.
Level of Evidence
Level IV, therapeutic study. See Instructions for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-013-3188-z
PMCID: PMC3792282  PMID: 23893361
2.  Wear of a 5 Megarad Cross-linked Polyethylene Liner: A 6-year RSA Study 
Background
One cross-linked polyethylene (XLPE) liner is manufactured using a lower dose of radiation, 5 Mrad, which may result in less cross-linking. The reported in vivo wear rate of this XLPE liner in patients undergoing THA has varied, and has included some patients in each reported cohort who had greater than 0.1 mm/year of wear, which is an historical threshold for osteolysis. Previous studies have measured wear on plain radiographs, an approach that has limited sensitivity.
Questions/purposes
We therefore measured the amount and direction of wear at 6 years using Radiostereometric analysis (RSA) in patients who had THAs that included a cross-linked polyethylene liner manufactured using 5 Mrad radiation.
Methods
We prospectively reviewed wear in 30 patients who underwent primary THAs with the same design of cross-linked acetabular liner and a 28-mm articulation. Tantalum markers were inserted during surgery and all patients had RSA radiographic examinations at 1 week, 6 months, 1, 2, and 6 years postoperatively.
Results
The mean proximal, two-dimensional (2-D) and three-dimensional (3-D) wear rates calculated between 1 year and 6 years were 0.014, 0.014, and 0.018 mm/per year, respectively. The direction of the head penetration recorded between 1 week and 6 years was in a proximal direction for all patients, proximolateral for 16 of 24 patients, and proximomedial for eight of 24 patients.
Conclusions
The proximal, 2-D and 3-D wear of a XLPE liner produced using 5 Mrad of radiation was low but measurable by RSA after 6 years. No patients had proximal 2-D or 3-D wear rates exceeding 0.1 mm/year. Further followup is needed to evaluate the effect of XLPE wear particles on the development of long-term osteolysis.
doi:10.1007/s11999-013-2789-x
PMCID: PMC3676600  PMID: 23334705
3.  Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction 
Autophagy  2011;7(12):1424-1433.
Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.
doi:10.4161/auto.7.12.18027
PMCID: PMC3327613
Atg13; autophagy; FIP200; Ulk1; Ulk2
4.  Identification of the Amino Acids 300–600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573 
PLoS ONE  2012;7(8):e43296.
Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573.
doi:10.1371/journal.pone.0043296
PMCID: PMC3422239  PMID: 22912850
5.  PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65 
Open Biology  2012;2(5):120080.
Summary
Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.
doi:10.1098/rsob.120080
PMCID: PMC3376738  PMID: 22724072
PINK1; Parkin; Parkinson's disease
6.  Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations 
Open biology  2011;1(3):110012.
Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro. We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated—namely Drosophila melanogaster (dPINK1), Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)—are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.
doi:10.1098/rsob.110012
PMCID: PMC3352081  PMID: 22645651
biochemistry; Parkinson's disease; kinase
7.  Second-generation Highly Cross-linked X3™ Polyethylene Wear: A Preliminary Radiostereometric Analysis Study 
Background
First-generation highly cross-linked polyethylene liners have reduced the incidence of wear particle-induced osteolysis. However, failed acetabular liners have shown evidence of surface cracking, mechanical failure, and oxidative damage. This has led to the development of second-generation highly cross-linked polyethylene, which has improved wear and mechanical properties and resistance to oxidation in vitro. Owing to its recent introduction, there are no publications describing its clinical performance.
Questions/purposes
We assessed early clinical wear of a second-generation highly cross-linked polyethylene liner and compared its clinical performance with the published results of hip simulator tests and with first-generation highly cross-linked polyethylene annealed liners.
Patients and Methods
Twenty-one patients were enrolled in a prospective cohort study. Clinical outcome and femoral head penetration were measured for 19 patients at 6 months and 1 and 2 years postoperatively.
Results
The median proximal head penetration was 0.009 mm and 0.024 mm at 1 and 2 years, respectively. The median two-dimensional (2-D) head penetration was 0.083 mm and 0.060 mm at 1 and 2 years, respectively. The median proximal wear rate between 1 and 2 years was 0.015 mm/year.
Conclusions
The wear rate calculated was similar to the in vitro wear rate reported for this material; however, it was less than the detection threshold for this technique. Although longer followup is required for wear to reach a clinically quantifiable level, this low level of wear is encouraging for the future clinical performance of this material.
Level of Evidence
Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-010-1259-y
PMCID: PMC3049610  PMID: 20151231
8.  Visualization and Biochemical Analyses of the Emerging Mammalian 14-3-3-Phosphoproteome* 
Molecular & Cellular Proteomics : MCP  2011;10(10):M110.005751.
Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease.
doi:10.1074/mcp.M110.005751
PMCID: PMC3205853  PMID: 21725060
9.  Reintervention after Mobile-bearing Oxford Unicompartmental Knee Arthroplasty 
Background
Medial compartment osteoarthritis is a common disorder that often is treated by unicompartmental knee arthroplasty (UKA). Although the Oxford 3 prosthesis is commonly used based on revision rate and cumulative survival, our experience suggests that although there may be adequate implant survival rates, we observed a worrisome and undisclosed reintervention rate of nonrevision procedures.
Purpose
We describe the frequency and cause of repeat intervention subsequent to implanting this device.
Methods
Between 1998 and 2005, 398 patients underwent UKA using the Oxford 3 prosthesis. The minimum followup was 12 months (mean, 43 months; range, 12–102 months).
Results
Forty of the 398 (10%) patients had 55 (13.8%) repeat anesthetics (reintervention). There were 38 nonrevision reinterventions. Revision was performed in 15 patients (3.8%), but two patients had a second revision (17 revisions or 4.3%). We revised the UKA to a second UKA in seven of the 15 cases but two subsequently were rerevised to a TKA; eight were revised directly to a TKA.
Conclusions
Although our data confirm the reported revision rates for this prosthesis, we observed a substantial reintervention rate. Most of the reinterventions are minor and are diagnosed frequently and treated arthroscopically. If revision is required, a second UKA may be considered and performed successfully in patients with isolated loosening of one component.
Level of Evidence
Level II, prognostic study. See Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-009-1089-y
PMCID: PMC2807008  PMID: 19768516
10.  Phosphorylation of Slx4 by Mec1 and Tel1 Regulates the Single-Strand Annealing Mode of DNA Repair in Budding Yeast▿  
Molecular and Cellular Biology  2007;27(18):6433-6445.
Budding yeast (Saccharomyces cerevisiae) Slx4 is essential for cell viability in the absence of the Sgs1 helicase and for recovery from DNA damage. Here we report that cells lacking Slx4 have difficulties in completing DNA synthesis during recovery from replisome stalling induced by the DNA alkylating agent methyl methanesulfonate (MMS). Although DNA synthesis restarts during recovery, cells are left with unreplicated gaps in the genome despite an increase in translesion synthesis. In this light, epistasis experiments show that SLX4 interacts with genes involved in error-free bypass of DNA lesions. Slx4 associates physically, in a mutually exclusive manner, with two structure-specific endonucleases, Rad1 and Slx1, but neither of these enzymes is required for Slx4 to promote resistance to MMS. However, Rad1-dependent DNA repair by single-strand annealing (SSA) requires Slx4. Strikingly, phosphorylation of Slx4 by the Mec1 and Tel1 kinases appears to be essential for SSA but not for cell viability in the absence of Sgs1 or for cellular resistance to MMS. These results indicate that Slx4 has multiple functions in responding to DNA damage and that a subset of these are regulated by Mec1/Tel1-dependent phosphorylation.
doi:10.1128/MCB.00135-07
PMCID: PMC2099619  PMID: 17636031
11.  Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress 
The Journal of Cell Biology  2007;176(1):89-100.
Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine–rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane–coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.
doi:10.1083/jcb.200605093
PMCID: PMC2063630  PMID: 17190791
12.  The LKB1-salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver 
Nature Communications  2014;5:4535.
LKB1 is a master kinase that regulates metabolism and growth through adenosine monophosphate-activated protein kinase (AMPK) and 12 other closely related kinases. Liver-specific ablation of LKB1 causes increased glucose production in hepatocytes in vitro and hyperglycaemia in fasting mice in vivo. Here we report that the salt-inducible kinases (SIK1, 2 and 3), members of the AMPK-related kinase family, play a key role as gluconeogenic suppressors downstream of LKB1 in the liver. The selective SIK inhibitor HG-9-91-01 promotes dephosphorylation of transcriptional co-activators CRTC2/3 resulting in enhanced gluconeogenic gene expression and glucose production in hepatocytes, an effect that is abolished when an HG-9-91-01-insensitive mutant SIK is introduced or LKB1 is ablated. Although SIK2 was proposed as a key regulator of insulin-mediated suppression of gluconeogenesis, we provide genetic evidence that liver-specific ablation of SIK2 alone has no effect on gluconeogenesis and insulin does not modulate SIK2 phosphorylation or activity. Collectively, we demonstrate that the LKB1–SIK pathway functions as a key gluconeogenic gatekeeper in the liver.
The liver is an important regulator of glucose homeostasis. Here, the authors provide insight into the molecular signalling pathways controlling hepatic gluconeogenesis by showing that SIK protein kinases suppress gluconeogenesis, and that glucagon—but not insulin—regulates phosphorylation of SIK2.
doi:10.1038/ncomms5535
PMCID: PMC4143937  PMID: 25088745

Results 1-12 (12)