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Background: The introduction of crosslinked ultra-high molecular weight polyethylene (XLPE) acetabular liners has been very successful, with decreased wear and reduction in the rates of revision hip arthroplasties. XLPE is the preferred articulation for most surgeons; however, there are concerns about the long-term performance of XLPE liners created with different manufacturing processes, which may lead to time-dependent failure, including accelerated wear, after several years.Questions/purposes: (1) What is the amount and rate of wear during the first 10 years using radiostereometric analysis (RSA) measurements of patients who had THAs that included a second-generation XLPE bearing? (2) Does the rate of wear change after 5 years in situ?Methods: This is a brief follow-up of a previous RSA study. In that study, we prospectively enrolled 21 patients with osteoarthritis who underwent primary cementless THA with an XLPE acetabular liner (three cycles of 3Mrad annealed) and 32-mm articulation. That group represented 44% of the 48 THAs performed by the surgeon at the hospital where RSA was available; 16 had cemented hips, leaving 32 who were invited to participate in this study. Of those, 11 lived rurally and declined to participate, leaving 21 patients who were included in the initial study. Since then, three patients died, one developed dementia and could not participate, and one had revision THA for reasons other than wear, leaving 16 patients available for analysis at 10 years. Tantalum markers were inserted during surgery, and all patients had RSA radiographs taken at 1 week, 6 months, and 1, 2, 5, and 10 years postoperatively. Femoral head penetration into the acetabular component was measured with RSA, including bedding-in during the first year and annual wear thereafter.Results: The median medial, proximal, anterior, two-dimensional (2D), and three-dimensional (3D) wear rates between 1 and 10 years were -0.001, 0.004, -0.012, 0.000, and 0.002 mm/year, respectively. No patient in this cohort had a proximal or 2D wear rate greater than 0.025 mm/year. The median proximal wear rate between 5 and 10 years (0.002 mm/year) was not greater than wear at 1 to 5 years (0.004 mm/year).Conclusion: Femoral head penetration in this second-generation XLPE liner remained very low at 10 years and accelerated wear after 5 years in situ did not occur. Concerns about late-onset wear from oxidation of irradiated-annealed XLPE were not observed. The low level of wear remains encouraging for the future clinical performance of this material.Level Of Evidence: Level II, therapeutic study. [ABSTRACT FROM AUTHOR]

Background: Initial stability of uncemented acetabular components in total hip arthroplasty (THA) is important for osseointegration and potentially enhanced by screw fixation. We used Australian Orthopaedic Association National Joint Replacement Registry data to determine whether screw usage influences uncemented acetabular component survival. Methods: Primary THA with uncemented acetabular components performed for osteoarthritis from 1999 to 2018 was included. Survivorship was calculated using Kaplan-Meier estimates of cumulative percent revision (CPR). Comparisons used Cox proportional hazards method. An instrumental variable analysis adjusted for surgeon preference for screws as a confounding factor was used. Results: Three hundred thirty thousand one hundred ninety-two THAs were included (31.8% with screws, 68.2% without). Two hundred twenty thousand six hundred seven were included in the instrumental variable analysis. Revision rate of acetabular components (all causes) was higher with screws during the first six years (hazard ratio (HR) = 1.45 (95% CI 1.34, 1.57), p < 0.001) and lower thereafter (HR = 0.81 (95% CI 0.67, 0.98), p = 0.027). Revision rate of acetabular components for loosening was higher with screws over the entire study period (HR = 1.73 (95% CI 1.51, 1.98), p < 0.001). Overall THA revision rate was higher with screws during the first six years (HR = 1.20 (95% CI 1.15, 1.26), p < 0.001) but lower thereafter (HR = 0.89 (95% CI 0.81, 0.98), p = 0.020). Revision rate for dislocation was higher with screws over the entire period (HR = 1.16 (95% CI 1.06, 1.26), p < 0.001). Instrumental variable analysis revealed higher revision rates with acetabular screws in the first six years. (HR = 1.18 (95% CI 1.09–1.29), p < 0.001). Conclusion: Screws did not confer a protective effect against acetabular loosening and were not associated with long-term negative consequences. [ABSTRACT FROM AUTHOR]

The author offers information on the a two days conference at the Duke University Law School where several judges, lawyers, and academics gathered to evaluate the state of civil litigation in federal court on May 2010 sponsored by the Advisory Committee on the Federal Rules of Civil Procedure.

The compound BAY 11-7082 inhibits I?Ba [inhibitor of NF- ?B (nuclear factor ?B)a] phosphorylation in cells and has been used to implicate the canonical IKKs (I?B kinases) and NF-?B in >350 publications. In the present study we report that BAY 11-7082 does not inhibit the IKKs, but suppresses their activation in LPS (lipopolysaccharide)-stimulated RAW macrophages and IL (interleukin)-1-stimulated IL-1R (IL-1 receptor) HEK (human embryonic kidney)-293 cells. BAY 11-7082 exerts these effects by inactivating the E2-conjugating enzymes Ubc (ubiquitin conjugating) 13 and UbcH7 and the E3 ligase LUBAC (linear ubiquitin assembly complex), thereby preventing the formation of Lys63-linked and linear polyubiquitin chains. BAY 11-7082 prevents ubiquitin conjugation to Ubc13 and UbcH7 by forming a covalent adduct with their reactive cysteine residues via Michael addition at the C3 atom of BAY 11-7082, followed by the release of 4-methylbenzene-sulfinic acid. BAY 11-7082 stimulated Lys48- linked polyubiquitin chain formation in cells and protected HIF1a (hypoxia-inducible factor 1a) from proteasomal degradation, suggesting that it inhibits the proteasome. The results of the present study indicate that the anti-inflammatory effects of BAY 11-7082, its ability to induce B-cell lymphoma and leukaemic T-cell death and to prevent the recruitment of proteins to sites of DNA damage are exerted via inhibition of components of the ubiquitin system and not by inhibiting NF-?B. [ABSTRACT FROM AUTHOR]

Abstract: ERK5 is a member of the mitogen-activated protein kinase (MAPK) family that, after stimulation, is activated selectively by dual phosphorylation in the TEY motif by MAPK kinase 5 (MEK5). ERK5 plays an important role in regulating cell proliferation, survival, differentiation and stress response. Moreover, it is involved in G2/M progression and timely mitotic entry. ERK5 is phosphorylated during mitosis, but the molecular mechanism by which it is regulated during this phase is still unclear. Here we show that although ERK5 is phosphorylated in mitosis, this does not occur on the activation motif (TEY), but at its C-terminal half. We have identified five sites of ERK5 phosphorylation in mitosis, two of them unknown. Furthermore, we demonstrate that ERK5 phosphorylation in mitosis is not MEK5-dependent, but rather, cyclin-dependent kinase (CDK)-dependent. Using a mutagenesis approach, we analysed the importance of the phosphorylated residues in ERK5 function; our evidence show that phosphorylation in mitosis of the residues identified inhibits ERK5 activity and regulates ERK5 shuttling from cytoplasm to the nucleus. These results reveal a previously unreported form of ERK5 regulation by phosphorylation and establish a link between CDK and ERK5 pathways during mitosis, which could be crucial for the correct progression of the cell cycle. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Store-operated calcium entry (SOCE) is an important Ca2+ entry pathway that regulates many cell functions. Upon store depletion, STIM1, a transmembrane protein located in the endoplasmic reticulum (ER), aggregates and relocates close to the plasma membrane (PM) where it activates store-operated calcium channels (SOCs). Although STIM1 was early defined as a phosphoprotein, the contribution of the phosphorylation has been elusive. In the present work, STIM1 was found to be a target of extracellular-signalregulated kinases 1 and 2 (ERK1/2) in vitro, and we have defined the ERK1/2-phosphorylated sites on the STIM1 sequence. Using HEK293 cells stably transfected for the expression of tagged STIM1, we found that alanine substitution mutants of ERK1/2 target sites reduced SOCE significantly, suggesting that phosphorylation of these residues are required to fully accomplish SOCE. Indeed, the ERK1/2 inhibitors PD184352 and PD0325901 decreased SOCE in transfected cells. Conversely, 12-O-tetradecanoylphorbol-13- acetate, which activates ERK1/2, enhanced SOCE in cells expressing wild-type tagged STIM1, but did not potentiate Ca2+ influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca2+ influx without disturbing the aggregation of STIM1 upon store depletion and without affecting the relocalization in ER-PM punctae. However, our results suggest that STIM1 phosphorylation at ERK1/2 target sites can modulate SOCE by altering STIM1 binding to SOCs, because a significant decrease in FRET efficiency was observed between alanine substitution mutants of STIM1-GFP and ORAI1-CFP. [ABSTRACT FROM AUTHOR]

Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression. [ABSTRACT FROM AUTHOR]

Background: Unicompartmental knee arthroplasty is a popular alternative to total knee replacement in selected patients. Component alignment has not yet been described by computer-assisted tomography (CAT) imaging techniques; these have been developed for total knee arthroplasty analysis. The aims of this study were to report two new technologies; a new unicompartmental knee arthroplasty system was radiographically assessed with a new CAT scan protocol. Methods: In a consecutive cohort study, 60 knees were analysed by the ‘UniCAT Protocol’. Patients were implanted with a unicompartmental knee arthroplasty system that uses a unique ligament tensor for femoral component alignment. The uniCAT protocol requires a long anteroposterior and lateral scout scan to measure limb alignment and component orientation. A spiral computer-assisted tomography at the knee is used to measure component rotation. The total scan time was 20 s with a calculated unshielded radiation dose of 1 mSv or less. Results: The mechanical axis had a mean of 2.7° varus. Femoral components were implanted with a mean of 0.37° valgus and 1.3° flexion. Tibial components were implanted with a mean 3.47° varus and 5.1° posterior slope. Femoral components were externally rotated a mean of 3.36°, tibial components were externally rotated 6.59° from the posterior tibia and 5.68° from the transepicondylar axis. Conclusion: The UniCAT protocol uses less radiation than whole-limb spiral scans and is a method that can be used with all modern computer-assisted tomography machines. The coronal and sagital alignment results compare favourably with previous published reports without computer-assisted tomography. Component rotation has not previously been reported and its implications are yet to be defined. [ABSTRACT FROM AUTHOR]

Background: Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers. Results: Here we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL. Conclusion: All three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death. [ABSTRACT FROM AUTHOR]

Phosphorylation regulates both normal and pathological Tau functioning. This microtubule-associated protein plays a role in the organization and integrity of the neuronal cytoskeleton under normal conditions and becomes hyperphosphorylated and aggregated in a number of neurodegenerative diseases referred to as tauopathies. In this study, we identify and compare the residues in human Tau phosphorylated in vitro by all four p38 MAPK isoforms, and study the regulation of the phosphorylation of Thr50, under conditions where p38 MAPKs are active in cells. Through biochemical analysis, loss of function studies and analysis of endogenous and overexpressed Tau proteins, we show that SAPK4/p38δ is the major kinase phosphorylating Thr50 in Tau, when cells are exposed to osmotic stress. We also show that mutation of Thr50 to glutamic acid, which mimics phosphorylation, increases the ability of Tau to promote tubulin polymerisation in vitro and in vivo. Moreover, we show that Thr50 is phosphorylated in filamentous Tau from Alzheimer's disease brain. These findings suggest a role for Tau in the adaptative response of neurons to stress and indicate that SAPK4/p38δ and/or SAPK3/p38γ may contribute to the hyperphosphorylation of Tau in the human tauopathies. [ABSTRACT FROM AUTHOR]

Presents a noninvasive ultrasound technique that may be used to differentiate complete and incomplete acute tears of the anterior talofibular ligament. Technique of ultrasound test; Demonstration of lesion through direct visualization; Confirmation upon operation.

An improved procedure has been developed for the isolation of insulin-stimulated protein kinase1 (ISPK-1), an S6 kinase-II homologue, by which 0.5 mg highly purified enzyme can be obtained within four days. The sequences of tryptic peptides from ISPK-I (100 residues) revealed 100% identity with the predicted protein product of rskmo-2, a cDNA clone isolated from a mouse F2 cell line library [Alcorta, D. A., Crews, C. M., Sweet, L. J., Bankston, L., Jones, S. W. and Erikson, R. L. (1989) Mol. Cell. Biol. 9, 3850-3859], demonstrating that rskmo-2 encodes an $6 kinase-II. Two isoforms of mitogen-activated protein (MAP) kinase (p42mapk and p44mapk) were the only ISPK-Ireactivating enzymes detected after Mono Q chromatography of extracts prepared from rabbit skeletal muscle or phaeochromocytoma 12 cells stimulated by nerve or epidermal growth factors. One of the residues on ISPK-I phosphorylated by p42mapk was a threonine located nine residues Nterminal to the conserved Ala-Pro-Glu motif in the C-terminal protein kinase domain, an analogous location to phosphorylation sites essential for the activity of cAMP-dependent protein kinase, MAP kinase and p34cdc2. A further threonine located five residues N-terminal to the same Ala-Pro-Glu motif was also phosphorylated, probably via autophosphorylation catalysed by ISPK-1 itself. [ABSTRACT FROM AUTHOR]

Improved methodology was used to establish that the phosphorylation of a serine located 10 residues from the N-terminus of glycogen synthase (N10) increases from 0.12 mol · mol-1 to 0.54 mol · mol-1 in vivo in response to adrenalin. The only ‘N10 kinase’ detected in muscle extracts was casein kinase-1 (CK1), although its activity was unaffected by injection of adrenalin in vivo or by incubation with cyclic-AMP-dependent protein kinase and MgATP in vitro. Prior phosphorylation of the serine residue N7 by phosphorylase kinase increased sixfold the rate of phosphorylation of glycogen synthase by CK1, and altered the specificity of CK1 so that it phosphorylated the serine residue N10 specifically. Stoichiometric phosphorylation of N7 decreased the activity ratio (± glucose 6-phosphate) of glycogen synthase from 0.80 to 0.45, and subsequent phosphorylation of N10 to 0.8 mol · mol-1 produced a further decrease to 0.17, demonstrating that N10 phosphorylation inhibits glycogen synthase. The major ‘N10 phosphatase’ in skeletal muscle extracts was identified as the glycogen-associated form of protein phosphatase-1 (PP1G), accounting for approximately 75% of the N10 phosphatase activity in the extracts and about 90% of the activity in isolated glycogen particles. Phosphorylation of N10, after prior phosphorylation of N7, decreased the rate of dephosphorylation of N7. These results, in conjunction with previous findings, establish that adrenalin inhibits glycogen synthase by increasing the phosphorylation of N7, N10 and three further serines located 30,4 and 38 residues from the start of the C-terminal CNBr peptide (termed the region C30-C38). They also indicate that increased phosphorylation of N10, the region C30-C38, and perhaps N7, is initiated through the inhibition of PP1G by adrenalin, which results from phosphorylation of its glycogen-targetting subunit by cyclic-AMP-dependent protein kinase [Hubbard, M. J. & Cohen, P. (1989) Eur. J. Biochem. 186, 711–716]. The conclusion that direct phosphorylation of glycogen synthase by cyclic-AMP-dependent protein kinase makes little contribution to inhibition by adrenalin, is at variance with the teachings of the major textbooks of biochemistry. [ABSTRACT FROM AUTHOR]

The amino acid sequence of glycogenin from rabbit skeletal muscle has been determined. The N-acetylated protein consists of 332 amino acids and has a molecular mass of 37278 Da. The novel tyrosyl-glucose linkage between glycogenin and glycogen [Smythe, C., Caudwell, F. B., Ferguson, M. & Cohen, P. (1988) EMBO J. 7, 2681 -2686] is shown to occur at a single site, tyrosine-194. Although glycogenin is a UDPaGIc utilising glucosyltransferase that self-glucosylates [Pitcher, J., Smythe, C. & Cohen, P. (1988) Eur. J. Biochem. 176, 391 - 395], following addition by an unknown enzyme of the first glucose to tyrosine-194, it is not homologous to either human glycogen synthase or other UDP-Glc-requiring enzymes. [ABSTRACT FROM AUTHOR]

Hormone-sensitive lipase is phosphorylated at a single site (site 2) in vitro by the AMP-activated protein kinase, without any direct effect on the activity of the enzyme. The amino acid sequence around this site has been determined. Ca2+/calmodulin-dependent protein kinase II also phosphorylates hormone-sensitive lipase predominantIy at this site, whilst cyclic-GMP-dependent protein kinase phosphorylates exclusively the regulatory site (site 1) which is also phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of site 2 has been found to inhibit subsequent phosphorylation and activation of hormone-sensitive lipase by the cyclic-AMPdependent and cyclic-GMP-dependent protein kinases, indicating that site-2 phosphorylation may have an antilipolytic role in vivo. [ABSTRACT FROM AUTHOR]

We have examined the sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P-labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the peptide containing the sites phosphorylated in vitro by casein kinase-2. The other may correspond to the 'I' site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme-bound phosphate (equivalent to <0.1 mol phosphate/mol 240-kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic-AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase respectively. We have sequenced peptides T4a and T4b from acetyl-CoA carboxylase derived from control and insulin-treated adipocytes, and also after phosphorylation in vitro with casein kinase-2 and the calmodulin-dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe-Ile-Ile-Gly-Ser4-Val-Ser5-Gln-Asp-Asn-Ser6 -Glu-Asp-Glu-Ile-Ser-Asn-Leu-. Site 5 was phosphorylated by the calmodulin-dependent protein kinase and site 6 by casein kinase-2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespjective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P-labelling of T4a. We were unable to isolate sufficient quantities of the other insulin-sensitive peptide to determine its sequence. Our results are consistent with the idea that insulin activates either casein kinase-2, or a protein kinase which has the same spiecificity as casein kinase-2. The function of this modification is not clear, since phosphorylation by casein kinase-2 has no direct effect on acetyl-CoA carboxylase activity. [ABSTRACT FROM AUTHOR]

We have examined the sites phosphorylated on acetyl-CoA carboxylase by three protein kinases which have been shown to inactivate the enzyme, i.e. cyclic-AMP-dependent protein kinase, acetyl-CoA carboxylase kinase-2 (ACK2, purified from rat mammary gland) and the AMP-activated protein kinase (formerly called acetyl-CoA carboxylase kinase-3, purified from rat liver). Each protein kinase phosphorylates two out of three sites (termed 1-3) which have been established by amino acid sequencing. The two sites phosphorylated by each kinase can be recovered on separate peptides, TC1 and TC2, derived by combined digestion of the native enzyme by trypsin and chymotrypsin: TC1 = Ser-²Ser(P)-Met-³Ser(P)-Gly-Leu; TC2 = Arg-Met-¹Ser(P)-Phe-Cyclic-AMP -dependent protein kinase phosphorylates sites 1 and 2 exclusively, whereas the AMP-activated protein kinase phosphorylates sites 1 and 3, plus at least one other minor site. ACK2 phosphorylates site 1 and, more slowly, an unidentified site(s) within TC1. We have also established the structures of the single major phosphopeptides (T1 and C1 respectively) which are recovered by HPLC after acetyl-CoA carboxylase phosphorylated by cyclic-AMP-dependent protein kinase is digested with trypsin or chymotrypsin alone. T1 is related to TC1, and has the structure: Ser-Ser(P)-Met-Ser-Gly-Leu-His-Leu-Val-Lys. C1 is identical with TC2. We have carried out studies on the correlation of the activity of acetyl-CoA carboxylase with the occupancy of sites 1, 2 and 3 during phosphorylation by each of the three protein kinases. The results suggest that phosphorylation of site 3 is primarily responsible for the large decrease in Vmax, produced by the AMP-activated protein kinase, while phosphorylation of site 1 may be primarily responsible for the increase in A0.5 for citrate and more modest depression of Vmax produced by cyclic-AMP-dependent protein kinase and ACK2. Our results emphasize that amino acid sequence information is essential in the unequivocal interpretation of data from phosphopeptide mapping experiments and allow a more complete interpretation of previous data on phosphorylation of acetyl-CoA carboxylase in intact cells. They also open the way to experiments which could establish the physiological roles of these protein kinases in the control of fatty acid synthesis. [ABSTRACT FROM AUTHOR]

Eukaryotic protein synthesis initiation factor 2 (eIF-2) can be phosphorylated on its a subunit by two well-characterised protein kinases, termed the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). Phosphorylation of eIF-2 by these kinases is thought to be important in the regulation of peptide-chain initiation. We report the location of the serine residue in the α subunit, which is phosphorylated by both these enzymes. Limited tryptic digestion and subsequent cyanogen bromide treatment of rat liver eIF-2 phosphorylated by HCR yielded one major phosphopeptide. This peptide had the sequence Ile-Leu-Leu-Ser-Glu-Leu-Ser(P)-Arg-Arg. The same major phosphopeptide was obtained from rabbit reticulocyte eIF-2 phosphorylated by HCR or dsI as judged by its behaviour on two-dimensional mapping and reverse-phase chromatography. In all cases the phosphorylated residue was found to be serine-7, and not serine-4, of the above sequence as determined front sequence analysis and by subdigestion of the peptide with Staphylococcus aureus V8 proteinase. [ABSTRACT FROM AUTHOR]

The complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-1, has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Straphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e.g. 1 - 49, 49 - 92, 67 -101, 108 - 134, 142 - 182 and 163 - 197) are inactive. Digestion with clostripain generated a peptide comprising residues 25-114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-1. [ABSTRACT FROM AUTHOR]

Storylines of Family Medicine is a 12-part series of thematically linked mini-essays with accompanying illustrations that explore the many dimensions of family medicine, as interpreted by individual family physicians and medical educators in the USA and elsewhere around the world. In 'IV: perspectives on practice--lenses of appreciation', authors address the following themes: 'Relational connections in the doctor-patient partnership', 'Feminism and family medicine', 'Positive family medicine', 'Mindful practice', 'The new, old ethics of family medicine', 'Public health, prevention and populations', 'Information mastery in family medicine' and 'Clinical courage.' May readers nurture their curiosity through these essays. [ABSTRACT FROM AUTHOR]

Background: Total knee replacement (TKR) has been shown to perform differently in patients with rheumatoid arthritis (RA) when compared to osteoarthritis (OA). In this study, we compare the survivorship between these two groups and examine patient and prosthesis factors that impact the revision rate. Methods: All RA and OA patients undergoing TKR in Australia from 1 September 1999 to 31 December 2016 were included. Revision rates were assessed using Kaplan–Meier estimates of survivorship. The cumulative percent revision analysed age, gender, prosthesis constraint and revision for infection. Results: There were 541 744 TKR procedures performed including 7542 patients with RA. RA declined as the primary diagnosis from 2.4% of all TKR in 2003 to 0.9% in 2016. Male sex was an independent revision risk in RA patients (hazard ratio (HR) = 1.66, P < 0.001) and OA patients (3.5 years+: HR = 1.09 (1.04–1.15), P < 0.001). Male RA patients had a higher revision rate for infection than females (HR = 3.14, P < 0.001). Females with RA had a lower cumulative percent revision compared to OA females, but males showed no difference between diagnoses. Revision in RA patients was not influenced by age. Compared to OA, RA patients had a decreased revision rate for those aged <65 years, but not for patients aged ≥65 years. Conclusion: The rate of revision after TKR in RA patients is lower than those with OA, but patients with RA are at increased risk of infection, particularly the male group. Prosthesis constraint had no influence on revision rate. Mortality in those undergoing TKR with RA was higher than in those with OA. This study shows that the use of primary total knee replacement (TKR) for rheumatoid arthritis (RA) is declining. The rate of revision after TKR in RA patients is lower than those with osteoarthritis, but patients with RA are at increased risk of infection, particularly the male group. For patients diagnosed with RA undergoing TKR, the rate of revision varies with gender but not with age. [ABSTRACT FROM AUTHOR]

The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations; PAK6 and PAK4 have fewer; and PAK4 is often amplified. To help interpret these genomic data, initially we compared the cellular regulation of the sister kinases and their roles in melanoma cells. In common with many ohnologue protein kinases, PAK4, PAK5 and PAK6 each have two 14-3-3-binding phosphosites of which phosphoSer99 is conserved. PAK4 localises to the leading edge of cells in response to phorbol ester-stimulated binding of 14-3-3 to phosphoSer99 and phosphoSer181, which are phosphorylated by two different PKCs or PKDs. These phosphorylations of PAK4 are essential for its phorbol ester-stimulated phosphorylation of downstream substrates. In contrast, 14-3-3 interacts with PAK5 in response to phorbol ester-stimulated phosphorylation of Ser99 and epidermal growth factor-stimulated phosphorylation of Ser288; whereas PAK6 docks onto 14-3-3 and is prevented from localising to cell-cell junctions when Ser133 is phosphorylated in response to cAMP-elevating agents via PKA and insulin-like growth factor 1 via PKB/Akt. Silencing of PAK4 impairs viability, migration and invasive behaviour of melanoma cells carrying BRAFV600E or NRASQ61K mutations. These defects are rescued by ectopic expression of PAK4, more so by a 14-3-3-binding deficient PAK4, and barely by PAK5 or PAK6. Together these genomic, biochemical and cellular data suggest that the oncogenic properties of PAK4 are regulated by PKC-PKD signalling in melanoma, while PAK5 and PAK6 are dispensable in this cancer. [ABSTRACT FROM AUTHOR]

Summary Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages. [ABSTRACT FROM AUTHOR]

We studied a prospective cohort of patients in whom gluteal tendon reconstruction was undertaken in association with hip arthroplasty. Over the course of 10 years, 24 patients had gluteal tendon reconstruction performed either at the time of hip arthroplasty or post-operatively, using the Ligament Augment and Reconstruction System (LARS), suture anchors, direct suture to bone, or a combination of these techniques. All patients were assessed clinically and by patient-centred outcome measures, including the hip disability and osteoarthritis score (HOOS). The mean post-operative HOOS was significantly better than pre-operative score (p < 0.05). The mean post operative score in the domains of symptoms, pain, activities of daily living (ADL), sports and quality of life (QoL) was 72 (SD 12.8), 73 (SD 15.9), 71 (SD 11.8), 54 (SD 22.6) and 57 (SD 21.76) respectively. There were two failures of gluteal tendon reconstruction which required revision using LARS. One patient died of an unrelated cause. Surgical intervention should be considered in gluteal tendinopathy at the time of hip arthroplasty or when symptoms occur following arthroplasty. [ABSTRACT FROM AUTHOR]

Background: The 'obesity epidemic' is expected to result in an increased incidence of knee osteoarthritis and hence total knee replacements (TKRs). Reviews have demonstrated the conflicting results of TKR for all obese (body mass index (BMI) >30). The aim of this literature review was to specifically evaluate outcomes of TKR in patients with morbid obesity (MO; BMI >40). Methods: A systematic review of medical databases (PubMed, Medline, Cochrane Library, ScienceDirect) by use of keywords from January 1990 to September 2009 was undertaken. Results: Clinical and functional Knee Society Scores (KSS) improve after TKR for patients with MO. The post-operative functional KSS was, in general, less than in controls. Radiographic analysis was inconclusive because of small study populations and short duration of follow-up. All studies reporting complications noted a greater prevalence in MO patients (10-30%). Of concern was the significantly higher prevalence of deep prosthetic infection (3-9-times that of controls). The morbidly obese also had a significantly higher incidence of wound complications. TKR did not result in weight loss for MO patients, and therefore has no benefit on weight-related medical conditions. Bariatric surgery in MO under 65 years of age has been shown to be a cost-effective and clinically effective method of weight reduction. This surgery also results in significant improvement in weight-related medical conditions, the KSS and knee pain. Conclusions: Given the increase in complications for MO patients after TKR, these patients should be advised to lose weight before surgery and, if suitable, would probably benefit from bariatric surgery. [ABSTRACT FROM AUTHOR]

The E3 ubiquitin ligase Pellino can be activated by phosphorylation in vitro, catalyzed by IL-1 receptor-associated kinase 1 (IRAK1) or IRAK4. Here, we show that phosphorylation enhances the E3 ligase activity of Pellino 1 similarly with any of several E2-conjugating enzymes (Ubc13-Uev1a, UbcH4, or UbcH5a/5b) and identify 7 amino acid residues in Pellino 1 whose phosphorylation is critical for activation. Five of these sites are clustered between residues 76 and 86 (Ser-76, Ser-78, Thr-80, Ser-82, and Thr-86) and decorate a region of antiparallel β-sheet, termed the "wing," which is an appendage of the forkhead-associated domain that is thought to interact with IRAK1. The other 2 sites are located at Thr-288 and Ser-293, just N-terminal to the RING-like domain that carries the E3 ligase activity. Unusually, the full activation of Pellino 1 can be achieved by phosphorylating any one of several different sites (Ser-76, Thr-86, Thr-288, or Ser-293) or a combination of other sites (Ser-78, Thr-80. and Ser-82). These observations imply that dephosphorylation of multiple sites is required to inactivate Pellino 1, which could be a device for prolonging Pellino's E3 ubiquitin ligase activity in vivo. [ABSTRACT FROM AUTHOR]

Background: Emerging longitudinal data appear to demonstrate an alarming trend towards an increasing prevalence of osteolysis-induced mechanical failure, following total knee replacement (TKR). Even with high-quality multi-plane X-rays, accurate pre-surgical evaluation of osteolytic lesions is often difficult. This is likely to have an impact on surgical management and provides reasonable indication for the development of a model allowing more reliable lesion assessment. The aim of this study, using a simulated cadaver model, was to explore the accuracy of rapid spiral computed tomography (CT) examination in the non-invasive evaluation of peri-prosthetic osteolytic lesions, secondary to TKR, and to compare this to conventional X-ray standards. Methods: A series of nine volume-occupying defects, simulating osteolytic lesions, were introduced into three human cadaveric knees, adjacent to the TKR implant components. With implants in situ, each knee was imaged using a two-stage conventional plain X-ray series and rapid-acquisition spiral CT. A beam-hardening artefact removal algorithm was employed to improve CT image quality. After random image sorting, 12 radiologists were independently shown the series of plain X-ray images and asked to note the presence, anatomic location and 'size' of osteolytic lesions observed. The same process was repeated separately for review of the CT images. The corresponding X-ray and CT responses were directly compared to elicit any difference in the ability to demonstrate the presence and size of osteolytic lesions. Results: Access to CT images significantly improved the accuracy of recognition of peri-prosthetic osteolytic lesions when compared to AP and lateral projections alone (P = 0.008) and with the addition of bi-planar oblique X-rays (P = 0.03). No advantage was obtained in accuracy of identification of such lesions through the introduction of the oblique images when compared with the AP and lateral projections alone (P = 0.13) Conclusion: The findings of this study suggest that peri-prosthetic osteolytic lesions can be reliably described non-invasively using a simple, rapid-acquisition CT-based imaging approach. The low sensitivity of conventional X-ray, even with provision of supplementary bi-planar 45° oblique views, suggests a limited role for use in situ for TKR implant screening where peri-prosthetic osteolytic lesions are clinically suspected. In contrast, the accuracy of CT evaluation, linked to its procedural ease and widespread availability, may provide a more accurate way of evaluating osteolysis around TKRs, at routine orthopaedic follow up. These findings have direct clinical relevance, as accurate early recognition and classification of such lesions influences the timing and aggressiveness of surgical and non-operative management strategies, and also the nature and appropriateness of planned implant revision or joint-salvaging osteotomy procedures. [ABSTRACT FROM AUTHOR]

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein-kinase (MAP) kinase-activated protein kinases 1 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphorylate bacterially expressed human tyrosine hydroxylase in vitro at comparable rates to other proteins thought to be physiological substrates of these protein kinases. The phosphorylation of all four alternatively spliced forms of human tyrosine hydroxylase by MAPKAP kinases-1 and -2 reached plateau values at 1 mol/mol subunit and 2 mol/mol subunit, respectively; the sites of phosphorylation were identified as Ser40 (MAPKAP kinase-1) and Ser19 and Ser40 (MAPKAP kinase-2). In contrast to calmodulin-dependent protein kinase-II, which phosphorylates Ser19 faster than Ser40, MAPKAP kinase-2 phosphorylated Ser40 about twice as fast as Ser19. The maximal activation of tyrosine hydroxylase by MAPKAP kinase-1 or-2 was about 3-fold, and activation by MAPKAP kinases-1 and -2 or calmodulin-dependent protein kinase-II correlated with the extent of phosphorylation of Ser40. The four alternatively spliced forms of human tyrosine hydroxylase were phosphorylated at Ser31 by MAP kinase, but at markedly different rates (3 = 4 > 1 > 2). Forms 3 and 4 were phosphorylated rapidly and stoichiometrically by MAP kinase doubling the activity, while phosphorylation of form 1 by MAP kinase to 0.4 mol/mol subunit increased activity by 40%. The effect on activity of phosphorylating both Ser31 and Ser40 was not additive. The possible roles of MAPKAP kinase-1, MAPKAP kinase-2 and MAP kinase in the regulation of tyrosine hydroxylase in vivo are discussed [ABSTRACT FROM AUTHOR]

Glycogen synthase from rabbit skeletal muscle has been shown to be a complex of two types of subunit which have apparent molecular masses of 86 kDa and 38 kDa and are present in a 1:1 molar ratio. The 38-kDa component was separated from the 86-kDa catalytic subunit by gel filtration in the presence of 2 M LiBr, and a number of chymotryptic peptides were sequenced. This demonstrated that the 38-kDa subunit was glycogenin, the protein that is bound covalently to glycogen and believed to be the 'primer' involved in the initiation of de novo glycogen synthesis. [ABSTRACT FROM AUTHOR]

Background: TKA generally has excellent long-term survivorship. When a new knee system supersedes a previous model, increased survivorship, improved functional performance, or both may be expected, because key areas of design modification are often targeted to address wear, stability, and the patellofemoral articulation. However, not all design changes are beneficial, and to our knowledge, knee arthroplasty has not been systematically evaluated in the context of design changes that occur during the development of new knee arthroplasty systems.Questions/purposes: Using the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) we performed multiple old-to-new comparisons of frequently used contemporary knee implants to ask: (1) does overall prosthesis survivorship free from revision increase when a new knee prosthesis system is introduced to replace a prior prosthesis system? (2) Has survivorship free from revision improved for the revision indications of wear, instability, and patellofemoral articulation issues, where development efforts have been concentrated?Methods: Data from the AOANJRR from September 1999 to December 2017 were used to compare the survivorship of prostheses free from revision at a maximum of 17 years in procedures where a new design model was introduced to replace a prior knee system from the same manufacturer. Only prosthesis systems used in a minimum of 2000 primary TKA procedures for osteoarthritis that had a minimum of 5 years of follow-up were included. Varus-valgus constrained and hinge TKA designs were excluded. Cruciate-retaining, posterior-stabilized, and medial pivot-design knees were considered separately. The new and old prosthesis systems were paired for analysis. Survivorship was calculated with Kaplan Meier estimates and comparisons were performed using the Cox proportional hazards method. Subanalyses according to the three main revision indications were performed, and where possible, analyses were performed based on polyethylene types (highly cross-linked polyethylene and ultra-high-molecular-weight polyethylene), combined and separated. Revision was defined as a reoperation of a previous knee arthroplasty in which one or more of the components was removed, replaced, or added. There were 323,955 TKA procedures and 11 new prosthesis system designs that were introduced to replace an earlier knee system from the same manufacturer. Of these prosthesis system pairs, six were cruciate-retaining prostheses, four were posterior-stabilized designs, and one was a medial pivot design.Results: Six of the 11 knee system pairs showed improved survivorship with the new design, three were no different, and in two, the newer prosthesis systems had a higher rate of revision than the old one did. When revision for wear was analyzed, five prosthesis systems showed improvement, five were no different, and one had a higher rate of revision than the previous system did. There was no improvement in the rate of revision for instability; seven new prosthesis systems showed no difference from the previous system and four new prosthesis systems had a higher rate of revision than the previous system did. A subanalysis of revision for patellofemoral complications showed improvement in two comparisons, no difference in six, and a higher revision rate in two; one could not be calculated because of an insufficient number of revisions for this reason.Conclusions: It is difficult to predict whether a new system will demonstrate better survival than a previous one, and widespread uptake of a new design before a benefit is shown in robust clinical studies is unwise. Similarly, adoption of a new system for which there is no difference in survivorship from a previous model may be premature because a new device may have associated unknown and unintended consequences. Healthcare policy makers and therapeutic device regulators should similarly be guided by results and seek out peer-reviewed evidence before accepting change to established practice. Surgeons must be aware that implant changes may not translate into better survivorship and must seek compelling evidence of improvement in survival and/or function before changing systems.Level Of Evidence: Level III, therapeutic study. [ABSTRACT FROM AUTHOR]

Pentosan polysulphate sodium (PPS) is a promising therapeutic agent for blocking knee pain in individuals with knee osteoarthritis (KOA). The mode of action of PPS in this context is unknown. We hypothesised that the osteocyte, being the principal cell type in the sub-chondral bone, was capable of expressing the pain mediator Nerve Growth Factor (NGF), and that this may be altered in the presence of PPS. We tested the expression of NGF and the response to PPS in the presence or absence of the proinflammatory cytokine tumour necrosis factor-alpha (TNFα), in human osteocytes. For this we differentiated human primary osteoblasts grown from subchondral bone obtained at primary knee arthroplasty for KOA to an osteocyte-like stage over 28d. We also tested NGF expression in fresh osteocytes obtained by sequential digestion from KOA bone and by immunofluorescence in KOA bone sections. We demonstrate for the first time the production and secretion of NGF/proNGF by this cell type derived from patients with KOA, implicating osteocytes in the pain response in this pathological condition and possibly others. PPS inhibited TNFα-induced levels of proNGF secretion and TNFα induced NGF mRNA expression. Together, this provides evidence that PPS may act to suppress the release of NGF in the subchondral bone to ameliorate pain associated with knee osteoarthritis. [ABSTRACT FROM AUTHOR]

Background: Community engagement is an important aspect of a successful rural placement. Materials and Methods: In this study, medical students undertaking longitudinal integrated clerkships at a rural clinical school instigated community engagement activities with a special school. Six health education sessions were delivered to eight adolescent special school students. This paper describes the perceptions of medical students and special school teachers in relation to the effect of this program on medical student personal and professional development, its acceptability by special school teachers, and the factors which contributed to the program outcomes. Two separate focus groups were conducted with seven medical students and two special school teachers. Results: Theme 1: Symbiotic nature of the program. There was perceived improvement in the medical students' communication, leadership and teaching skills, and their understanding of working with people with disabilities. Special school teachers noted benefits to their students from the health expertise and role modelling provided. The university experienced enhanced links with the community. Theme 2: Factors that contributed to the success of this community engagement activity. All parties wanted to engage in the program. Valuable time was spent developing relationships and preparing with all stakeholders. Constructive teamwork was paramount. Discussion: Involvement in this program gave students a unique opportunity to develop skills in professionalism that are essential to working as health practitioners but difficult for universities to teach. The voluntary nature of the initiative was novel, promoting this skill development and enhancing the effectiveness of the program. The factors that contributed to the success of this program are potentially applicable to other settings. Conclusion: This initiative was highly acceptable to the special school teachers involved and was perceived to have positive effects on medical student personal and professional development. [ABSTRACT FROM AUTHOR]

ABSTRACT A number of different software programs are used to investigate the in vivo wear of polyethylene bearings in total hip arthroplasty. With wear rates below 0.1 mm/year now commonly being reported for highly cross-linked polyethylene (XLPE) components, it is important to identify the accuracy of the methods used to measure such small movements. The aims of this study were to compare the accuracy of current software programs used to measure two-dimensional (2D) femoral head penetration (FHP) and to determine whether the accuracy is influenced by larger femoral heads or by different methods of representing the acetabular component within radiostereometric analysis (RSA). A hip phantom was used to compare known movements of the femoral head within a metal-backed acetabular component to FHP measured radiographically using RSA, Hip Analysis Suite (HAS), PolyWare, Ein Bild Roentgen Analyse (EBRA), and Roentgen Monographic Analysis Tool (ROMAN). RSA was significantly more accurate than the HAS, PolyWare, and ROMAN methods when measuring 2D FHP with a 28 mm femoral head. Femoral head size influenced the accuracy of HAS and ROMAN 2D FHP measurements, EBRA proximal measurements, and RSA measurements in the proximal and anterior direction. The use of different acetabular reference segments did not influence accuracy of RSA measurements. The superior accuracy and reduced variability of RSA wear measurements allow much smaller cohorts to be used in RSA clinical wear studies than those utilizing other software programs. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:988-996, 2017. [ABSTRACT FROM AUTHOR]

Osteocytes are essential regulators of bone homeostasis. However, they are difficult to study due to their location within the bone mineralised matrix. Although several techniques have been published for the isolation of osteocytes from mouse bone, no such technique has been described for human osteocytes. We have therefore developed a protocol for the isolation of osteocytes from human trabecular bone samples acquired during surgery. The cells were digested from the bone matrix by sequential collagenase and ethylenediaminetetraacetic acid (EDTA) digestions and the cells from later digests displayed characteristic dendritic osteocyte morphology when cultured ex vivo . Furthermore, the cells expressed characteristic osteocyte marker genes, such as E11 , dentin matrix protein 1 ( DMP1 ), SOST , matrix extracellular phosphoglycoprotein ( MEPE ) and phosphate regulating endopeptidase homologue, X-linked ( PHEX ). In addition, genes associated with osteocyte perilacunar remodelling, including matrix metallopeptidase-13 ( MMP13 ), cathepsin K ( CTSK ) and carbonic anhydrase 2 ( CAR2 ) were expressed. The cells also responded to parathyroid hormone (PTH) by downregulating SOST mRNA expression and to 1α,25-dihydroxyvitamin D 3 (1,25D) by upregulating fibroblast growth factor 23 ( FGF23 ) mRNA expression. Therefore, the cells behave in a similar manner to osteocytes in vivo . These cells represent an important tool in enhancing current knowledge in human osteocyte biology. [ABSTRACT FROM AUTHOR]

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. [ABSTRACT FROM AUTHOR]

Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser858 of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease. [ABSTRACT FROM AUTHOR]

Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser111) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease. [ABSTRACT FROM AUTHOR]

Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation ( ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process. [ABSTRACT FROM AUTHOR]

Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys33-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6-AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked Ub chains. Intriguingly, the crystal structure of Lys33-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys11- linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type. [ABSTRACT FROM AUTHOR]

Background and purpose - Wear rates of highly crosslinked polyethylene (XLPE) acetabular components have varied considerably between different published studies. This variation is in part due to the different techniques used to measure wear and to the errors inherent in measuring the relatively low amounts of wear in XLPE bearings. We undertook a scoping review of studies that have examined the in vivo wear of XLPE acetabular components using the most sensitive method available, radiostereometric analysis (RSA). Methods - A systematic search of the PubMed, Scopus, and Cochrane databases was performed to identify published studies in which RSA was used to measure wear of XLPE components in primary total hip arthroplasty (THA). Results - 18 publications examined 12 primary THA cohorts, comprising only 260 THAs at 2-10 years of follow-up. The mean or median proximal wear rate reported ranged from 0.00 to 0.06 mm/year. However, differences in the manner in which wear was determined made it difficult to compare some studies. Furthermore, differences in RSA methodology between studies, such as the use of supine or standing radiographs and the use of beaded or unbeaded reference segments, may limit future meta-analyses examining the effect of patient and implant variables on wear rates. Interpretation - This scoping review confirmed the low wear rates of XLPE in THA, as measured by RSA. We make recommendations to enhance the standardization of reporting of RSA wear results, which will facilitate early identification of poorly performing implants and enable a better understanding of the effects of surgical and patient factors on wear. [ABSTRACT FROM AUTHOR]

Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau-tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with aphosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis. [ABSTRACT FROM AUTHOR]

Salt-inducible kinase 2 (SIK2) is an AMP-activated protein kinase (AMPK) related kinase abundantly expressed in adipose tissue. Our aim was to identify molecular targets and functions of SIK2 in adipocytes, and to address the role of PKA-mediated phosphorylation of SIK2 on Ser358. Modulation of SIK2 in adipocytes resulted in altered phosphorylation of CREB-regulated transcription co-activator 2 (CRTC2), CRTC3 and class IIa histone deacetylase 4 (HDAC4). Furthermore, CRTC2, CRTC3, HDAC4 and protein phosphatase 2A (PP2A) interacted with SIK2, and the binding of CRTCs and PP2A to wild-type but not Ser358Ala SIK2, was reduced by cAMP elevation. Silencing of SIK2 resulted in reduced GLUT4 (also known as SLC2A4) protein levels, whereas cells treated with CRTC2 or HDAC4 siRNA displayed increased levels of GLUT4. Overexpression or pharmacological inhibition of SIK2 resulted in increased and decreased glucose uptake, respectively. We also describe a SIK2-CRTC2-HDAC4 pathway and its regulation in human adipocytes, strengthening the physiological relevance of our findings. Collectively, we demonstrate that SIK2 acts directly on CRTC2, CRTC3 and HDAC4, and that the cAMP-PKA pathway reduces the interaction ofSIK2 with CRTCs and PP2A. Downstream, SIK2 increases GLUT4 levels and glucose uptake in adipocytes. [ABSTRACT FROM AUTHOR]

Abstract: Insulin and muscle contraction each stimulate translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle, an important process regulating whole-body glucose homeostasis. RalA mediates insulin-stimulated GLUT4 translocation; however, it is unclear how this small GTPase is regulated in skeletal muscle in response to insulin. Here, we identified GARNL1/RalGAPα1, a major α subunit of the Ral-GTPase activating protein in skeletal muscle, as a protein whose phosphorylation and binding to the regulatory 14-3-3 proteins is stimulated by insulin and also by muscle contraction. The insulin-stimulated interaction with 14-3-3 involved PKB/Akt-mediated phosphorylation of Thr735 on GARNL1/RalGAPα1. Knockdown of GARNL1/RalGAPα1 increased, while overexpression of GARNL1/RalGAPα1Thr735Ala mutant protein decreased, the RalA activation and the RalA-dependent GLUT4 translocation in response to insulin in muscle cells. These findings show that GARNL1/RalGAPα1 is the missing link that connects the insulin-PKB/Akt signaling pathway with the activation of the RalA small GTPase in muscle cells. GARNL1/RalGAPα1 and its phosphorylation and/or binding to 14-3-3s are critical for GLUT4 trafficking through RalA in muscle cells. [Copyright &y& Elsevier]

NUAK1 (NUAK family SnF1-like kinase-1) and NUAK2 protein kinases are activated by the LKB1 tumour suppressor and have been implicated in regulating multiple processes such as cell survival, senescence, adhesion and polarity. In the present paper we present evidence that expression of NUAK1 is controlled by CDK (cyclin-dependent kinase), PLK (Polo kinase) and the SCFβTrCP (Skp, Cullin and F-boxβTrCP) E3 ubiquitin ligase complex. Our data indicate that CDK phosphorylates NUAK1 at Ser445, triggering binding to PLK, which subsequently phosphorylates NUAK1 at two conserved non-catalytic serine residues (Ser476 and Ser480). This induces binding of NUAK1 to βTrCP, the substrate-recognition subunit of the SCFβTrCP E3 ligase, resulting in NUAK1 becoming ubiquitylated and degraded. We also show that NUAK1 and PLK1 are reciprocally controlled in the cell cycle. In G2–M-phase, when PLK1 is most active, NUAK1 levels are low and vice versa in S-phase, when PLK1 expression is low, NUAK1 is more highly expressed. Moreover, NUAK1 inhibitors (WZ4003 or HTH-01-015) suppress proliferation by reducing the population of cells in S-phase and mitosis, an effect that can be rescued by overexpression of a NUAK1 mutant in which Ser476 and Ser480 are mutated to alanine. Finally, previous work has suggested that NUAK1 phosphorylates and inhibits PP1βMYPT1 (where PP1 is protein phosphatase 1) and that a major role for the PP1βMYPT1 complex is to inhibit PLK1 by dephosphorylating its T-loop (Thr210). We demonstrate that activation of NUAK1 leads to a striking increase in phosphorylation of PLK1 at Thr210, an effect that is suppressed by NUAK1 inhibitors. Our data link NUAK1 to important cell-cycle signalling components (CDK, PLK and SCFβTrCP) and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1βMYPT1 phosphatase. [ABSTRACT FROM AUTHOR]