The purpose of this cohort study was to assess the incidence of positive cultures in section’s osseous slice biopsy (SOB) taken at the level of major limb amputation. In case of positive cultures we sought whether the microorganisms present in SOB could take origin from the primary infection site necessitating the amputation. The impact of diabetes on culture results was also investigated.P < 0.05.This prospective cohort study, which aimed to confirm the results of the pilot study, analysed patients who underwent major limb amputation between 2012 and 2013 in three Lithuanian hospitals. SOBs at the amputation site  and percutaneous bone biopsies of the distal site were performed simultaneously during limb amputation. Tissue cultures were analysed by microbiologists, and species along with antibiograms were reported. Histopathological assessment and bacterial typing were also evaluated. A positive culture was defined as the identification of at least 1 bacteria not belonging to the skin flora, at least 2 bacteria belonging to the skin flora with the same antibiotic susceptibility profiles or the same bacteria belonging to the skin flora in two different sites. Fisher’s exact test and Student’s test were used to compare the populations and the microbiological results. The statistical significance level was set at Sixty-nine patients , mean age 68.7 (S = 13.6) years, including 21 (30.4 %) with diabetes underwent the major limb amputation. Forty-five amputations (65.2 %) were done above the knee. In total, 207 SOBs and 207 percutaneous distal site biopsies were studied. SOB cultures were positive in 11 (15.9 %) cases. In 5 (45.5 %) cases the same microorganisms were identified in both SOB and distal biopsy cultures. No association between culture results and presence of diabetes was identified.Our results suggest that, independently of the diabetes status, foot infection may silently spread along the bone and can achieve the site of major limb amputation. Additional investigations aiming to confirm this hypothesis and to evaluate a prognostic value are in progress. Section’s osseous slice biopsy (SOB) is rarely performed in routine amputations, partially due to additional cost and the threat of increased risk of adverse events. However, the belief that the proximal amputation site distant from the site necessitating the limb amputation is unlikely to be involved in the infectious process, is still frequent and this may explain why SOB is regularly omitted from the amputation site selection process. Data about the probability of the extension of the infectious process affecting the distal site responsible for the amputation, prognostic value are lacking and findings present have only been established by taking superficial microbiology cultures.We hypothesized that infection from the distal site may spread along the bone and be present at the amputation site. Therefore, a positive SOB culture could be a risk factor for delayed stump healing. Our team has previously participated in a retrospective pilot study in France, which compared the culture results of SOB with those of bone biopsy at the initial distal infection site . The resThis is a 1 year follow-up cohort study. Stump wound healing, postoperative antibiotic therapy will be assessed at the secondary end point of the study.In this preliminary study we report the incidence of positive cultures in section’s osseous slice biopsy (SOB) taken at the level of major limb amputation and compare the microbiology of positive cultures with percutaneous bone biopsy cultures performed at distal infected site. We also analyze the relationship between diabetes and microbiological culture results.In this multicenter prospective cohort study we included adult patients who underwent a major amputation of lower limb in the Hospital of Lithuanian University of Health Sciences Kauno klinikos, Kaunas Clinical Hospital and Republican Hospital of Kaunas between 2012 and 2013. The only exclusion criterion was the refusal to participate in the study. Patients had a complete medical history taken, full examination and tests performed. The following data were collected: age, sex, diabetes status and glycemic control, presence of peripheral vascular disease, osteoarthritis, level of infection, context of trauma, level of amputation, immunosuppression, bedridden disability, comorbidity, duration and history of the pathology, reason of the amputation, time from the decision to amputate to surgical procedure, history of previous amputations, total duration of antibiotic therapy before the amputation, antibiotic-free interval before amputation, inflammatory markers (C-reactive protein), renal function , and antibiotic use before and after hospitalization.Peripheral vascular disease was assumed to be present if 1) dorsal-pedal and posterior-tibial pulses were absent and/or 2) pathological results were evident on Doppler UltraSound, peripheral arteriography and/or 3) there was a previously made diagnosis of peripheral vascular disease by vascular surgeon by using the methods mentioned above. Comorbidity status was evaluated on the presence of cardiac, renal or hepatic insufficiency. Patients were considered to have had an antibiotic-free interval, if they had not received any systemic antibiotic for at least 2 weeks before the amputation. An informed consent form had to be completed by every patient who met the inclusion criteria. The study protocol was approved by Lithuanian Ethics Committee .Concomitant SOB and percutaneous bone biopsy of the distal site were performed during limb amputation. A new surgical setup and new instruments were used, to try and decrease the likelihood of cross-contamination during surgery. Percutaneous bone biopsy was performed in the surgical room using an 11-gauge biopsy needle inserted through a 5–10-mm skin incision at least 20 mm from the ulcer periphery, to avoid contamination by the colonizing flora, following the methodology described in the literature [2 and Mac Conkey agar plates – at 35 °C for 18–24 h. If culture was negative at the first observation, sheep blood and chocolate agar plates were re-examined after a second 24 h incubation. Thioglycollate broth was incubated at 35 °C temperature for 5 days. Schaedler agar was incubated at 35 °C in an anaerobic workstation Bug Box (UK) for 2 weeks. Maldi-TOF-MS (BRUKER) mass spectrometry was used for microorganism identification. Analysis of the number of colony-forming units per culture was performed by means of a counting frame for bone biopsy cultures. A positive culture was defined as the identification of at least 1 bacteria not belonging to the skin flora, at least 2 bacteria belonging to the skin flora (CoNS (coagulase negative staphylococci), Corynebacterium spp) with the same antibiotic susceptibility profiles or the same bacteria belonging to the skin flora in two different sites. A doubtful culture was defined as the identification of one bacteria belonging to the skin flora in one site. The dominant pathogen required at least a three times higher growth in bone cultures than other microorganisms. All positive cultures were frozen at −60 °C for further research. The isolated pathogens from SOB and distal site biopsy, which happened to be the same species were compared using genotyping (pulsed field gel electrophoresis) to confirm the same strain.Specimens were immediately placed into Amies transport medium , brought to the microbiology laboratory within 1 h after sampling. The samples were inoculated directly into 5 % sheep blood agar , chocolate agar , Mac Conkey agar plates , Schaedler agar  and thioglycollate broth. Sheep blood and chocolate agar plates were incubated at 35 °C in an atmosphere containing 5 % COBone fragment was fixed in 4 % paraformaldehyde, decalcified in 10 % HCl and embedded in paraffin for histological analysis. 3-μm thick sections were made, stained with hematoxylin-eosin.Histopathological findings for the signs of infection were evaluated. Histopathological findings in bone specimens were defined as acute osteomyelitis when necrosis, destroyed bone and infiltrations of polymorphonuclear granulocytes at cortical sites and inside the bone marrow were present. Congestion or thrombosis of medullary or periosteal small vessels was also a frequent finding. It was defined as chronic osteomyelitis when there was destroyed bone and infiltrations of lymphocytes, histiocytes and/or plasmatic cells at cortical sites and inside the bone marrow. All cases of osteomyelitis exhibited areas of fibrosis in variable forms and medullar oedema.P < 0.05.Fisher’s exact test and Student’s test were used to compare the populations and microbiological results, all the variables had normal distribution. The statistical significance level was set at From 2012 to 2013, 69 patients ) underwent major limb amputation at Kaunas hospitals. Among all these 69 patients, 45 (65.2 %) had above the knee amputation and 21 (30.4 %) were known to have diabetes (Table In 5 (45.5 %) cases the same microorganisms  were identified in SOB and distal site bone biopsy cultures Fig. . No specSOB cultures were positive in 5 23.8 %) patients suffering from diabetes and in 6 (12.5 %) patients without diabetes (p = 0.29). Microorganisms identified in SOB were also cultured from the distal site in 1 (20 %) diabetic and 4 (66.7 %) non-diabetics (p = 0.24) . SOB cultures were positive in 42.1 % of the pilot study patients, while the microorganism at amputation and distal sites coincided in 69.6 % of the cases [. SOB culp < 0.001) [et al. presented a series of cases in which S. epidermidis was isolated from the bone biopsy and the histopathological studies, confirming diagnosis of osteomyelitis, but no anaerobic cultures were performed [In previous studies, the rate CoNS isolation from bone tissue varied between 10 % and 50 %, and was considered in most cases as true infections , 7, 8. L< 0.001) . This fi< 0.001) . Nevertherformed . In the et al. study with quantitative cultures, coagulase negative staphylococci were considered as commensals, since their quantification was inferior to other concomitant and more classical pathogens of osteomyelitis [Differentiating the true coagulase-negative staphylococcal infection from contamination has an important impact on therapeutic implications. In this situation, clinical, radiological findings, as well as histological signs of infection may be useful. In our study only in 2 cases the signs of infection were found in histopathological assessement of SOB. We think that the microorganisms spread and were found in SOB earlier until the changes are visible in histopathological or radiological analysis. Repeated positive cultures, isolation of identical species with comparable antibiotic susceptibility profile were considered as strong arguments in favour of a true infection in recent study . In thismyelitis . Of notemyelitis –14.To confirm our hypothesis that culture results of the slice site are reliable and bacteria ascend from the foot instead of from skin, blood, or other nearby source of infection and affect the amputation outcome, further investigations and analysis are in progress.Our results suggest that, independently of the diabetes status, foot infection may silently spread along the bone and can achieve the site of major limb amputation. Additional investigations aiming to confirm this hypothesis and to evaluate a prognostic value are in progress.


The mammalian/mechanistic target of rapamycin (mTOR) exists in two complexes, mTORC1 and mTORC2. Both mTORC1 and mTORC2 contain the mTOR kinase and three other common components: mLST8, deptor, and Tti1/Tel. Additionally, mTORC1 contains PRAS40 and raptor, while mTORC2 contains mSIN1, protor, and rictor [UBE3A gene as the cause for AS [Our recent studies have revealed that mTOR signaling is abnormal in the cerebellum of the mouse model for Angelman syndrome (AS) . AS is ae for AS , 5. In oe for AS . We foune for AS . Thus, re for AS , indicate for AS . Anothere for AS . These rOur results also raise additional questions. For instance, it is well known that mTORC2 activates mTORC1 through AKT phosphorylation, yet our results showed mTORC1 overactivation, despite reduced mTORC2-AKT activation. Could this runaway mTORC1 activation due to an ongoing autophosphorylation? Are TSC2 inhibition or other unidentified factors involved? We showed that mTORC2 inhibition was due to mTORC1S6K1-mediated inhibitory phosphorylation of rictor and possibly TSC2 as well. It remains to be determined how general this regulation is in different brain regions, and whether additional mutual interactions occur at different levels of mTOR signaling cascades. Finally, it is noteworthy that mTOR activity is increased in Fragile X, tuberous sclerosis, and Down's syndromes, but decreased in Rett's syndrome . However, in all these variants of autism syndrome disorders, it is not clear whether alterations in mTOR signaling are due to modifications of mTORC1, mTORC2, or both. Further studies directed at these issues should provide a better understanding of these developmental neurologic disorders and reveal potential new therapeutic targets.


The mechanism by which the drug rapamycin inhibits the mechanistic target of rapamycin (mTOR) is of intense interest because of its likely relevance in cancer biology, aging, and other age-related diseases. While rapamycin acutely and directly inhibits mTORC1, only chronic administration of rapamycin can inhibit mTORC2 in some, but not all, cell lines or tissues. The mechanism leading to cell specificity of mTORC2 inhibition by rapamycin is not understood and is especially important because many of the negative metabolic side effects of rapamycin, reported in mouse studies and human clinical trials, have been attributed recently to mTORC2 inhibition. Here, we identify the expression level of different FK506-binding proteins (FKBPs), primarily FKBP12 and FKBP51, as the key determinants for rapamycin-mediated inhibition of mTORC2. In support, enforced reduction of FKBP12 completely converts a cell line that is sensitive to mTORC2 inhibition to an insensitive cell line, and increased expression can enhance mTORC2 inhibition. Further reduction of FKBP12 in cell lines with already low FKBP12 levels completely blocks mTORC1 inhibition by rapamycin, indicating that relative FKBP12 levels are critical for both mTORC1 and mTORC2 inhibition, but at different levels. In contrast, reduction of FKBP51 renders cells more sensitive to mTORC2 inhibition. Our findings reveal that the expression of FKBP12 and FKBP51 is the rate limiting factor that determines the responsiveness of a cell line or tissue to rapamycin. These findings have implications for treating specific diseases, including neurodegeneration and cancer, as well as targeting aging in general. Interin vivo, but the effects of rapamycin on mTORC2 in individual tissues have not been clearly established and is critical to understand the effects of rapamycin in vivo. To address this issue, we IP injected 10-week-old mice with 8 mg kg−1 rapamycin every other day for 3 weeks. Mice were fasted overnight and treated with insulin for 15 min prior to tissue harvest, as described by Lamming et al.  or an overnight fast with 15-min insulin stimulation , further supporting the hypothesis that FKBP12 is required for inhibition of mTORC2 in vivo.To test whether the differential response of the various tissues to mTORC2 inhibition by rapamycin is due to altered levels of FKBPs, we examined their expression in multiple tissues. FKBP12 and FKBP51 levels have been reported in human tissues . As we have demonstrated that the level of FKBP12 is critical to cellular responsiveness to mTORC2 inhibition by rapamycin, we postulate that it will be imperative to obtain similar expression levels of functional FBKP12 to that in PC3 cells to sensitive HeLa cells to mTORC2 inhibition.FKBP51−/− MEFs are more sensitive to mTORC2 inhibition, but Rictor dissociation is not directly affected, indicating that FKBP51 indirectly affects mTORC2 complex formation. Instead FKBP51−/− MEFs have impaired inhibition of mTORC1, which may in turn be affecting mTORC2 activity either through feedback regulation by the mTORC1 pathway on mTORC2 or simply through competing with FKBP12 for rapamycin. These results point to a model where FBKP12 and FKBP51 are required for the direct inhibition of mTORC1, while FKBP12 is the only FKBP which can bind free mTOR leading to inhibition of both mTORC1 and mTORC2 after prolonged exposure to the drug . All cell lines were cultured at 37 °C under an atmosphere of 95% air and 5% CO2. Following treatments, cells were harvested in RIPA buffer  and protein concentrations were determined using the DC protein assay .PC3 cells were maintained in F12K media supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 met al., −1) before experimentation.Lentiviral pGIPZ shRNA constructs were purchased from Thermo Scientific . Briefly, HEK 293T cells were transfected with pGIPZ constructs bearing shRNA against FKBP12 or FKBP51 or a nonspecific control using calcium phosphate transfection as previously described . Western blot analysis was performed using the following protein-/phospho-protein-specific antibodies: anti-p70 S6 kinase (#2708), anti-phospho-p70 S6 kinase (T389) (#9234), anti-rpS6 (#2217), anti-phospho-rpS6(S240/244) (#5364), anti-phospho-NDRG-1 (T346) (#5482), anti-NDRG-1 (#9485), anti-phospho-Akt (S473) (#4060), anti-Akt (#4691), anti-FKBP51 (#12210), anti-FKBP52 (#11826), anti-mTOR (#2972), anti-Rictor (#9476), anti-phospho-Rictor (T1135) (#3806), and anti-Raptor (#2280) were from Cell Signaling Technologies . Anti-FKBP25 (ab16654) and anti-FKBP12 (ab2918) were from Abcam. Anti-GAPDH (AM4300) was from Ambion Austin, TX.m Hepes, pH 7.5, 120 mm NaCl, 1 mm EDTA, 0.3% CHAPS, 10 mm pyrophosphate, 10 mm β-glycerophosphate, 50 mm NaF, 0.5 mm orthovanadate, protease inhibitor tablet) as described  or vehicle every other day for 10 days (4–5 mice/group). Mice were fasted overnight (16 h) following the last rapamycin injection and injected with insulin for 15 min just before tissue harvesting. Tissues were immediately frozen in liquid nitrogen. Alternatively, tissues were dissected from mice that were fasted for 6 h and immediately frozen in liquid nitrogen. The tissues were homogenized using the Omni TH homogenizer  on ice in RIPA buffer  and then centrifuged at 21,130 × g for 15 min at 4 °C. The supernatants were collected, and protein concentration was determined using the DC protein assay (Bio-Rad).Ten-week-old C57BL/6J mice were given intraperitoneal injections of 8 mg kg


To study this high heterogeneity in the inhibitory effects of NSAIDs, we have developed an in silico approach to evaluate NSAID action on targets under different PGHS-1 microenvironmental conditions, such as arachidonic acid, reducing cofactor, and peroxide concentrations. The designed technique permits translating the drug IC50, obtained in one experimental setting to another, and predicts in vivo inhibitory effects based on the relevant in vitro data. For the aspirin case, we elucidated the mechanism underlying the enhancement and reduction (aspirin resistance) of its efficacy, depending on PGHS-1 microenvironment in in vitro/in vivo experimental settings. We also present the results of the in silico screening of the combined action of sets of two NSAIDs , and study the mechanism of the experimentally observed effect of the suppression of aspirin-mediated PGHS-1 inhibition by selective and nonselective NSAIDs. Furthermore, we discuss the applications of the obtained results to the problems of standardization of NSAID test assay, dependence of the NSAID efficacy on cellular environment of PGHS-1, drug resistance, and NSAID combination therapy.The detailed kinetic model of Prostaglandin H Synthase-1 (PGHS-1) was applied to  The E2 state can be converted back to the resting state E1 by two consecutive reactions with an external reducing cosubstrate, RC, with a release of oxidised cosubstrate, OC (reactions 10 and 12). Alternatively, the E2 state can undergo a one-electron internal reduction via intramolecular electron transfer from the tyrosine 385 residue (Tyr385) to a protoporphyrin group (reaction 13), resulting in the formation of the E5 state,  containing the Tyr385* radical and a ferryl heme (Intermediate II). The E5 state can also complete the POX cycle and return to the resting state E1 via reactions 9 and 10 with cosubstrate. According to the Branched Chain Mechanism, the presence of the Tyr385* radical is ultimately required for the activation of AA oxidation reaction in the COX-site  (E9), containing AA in the COX-site. Reaction 2 corresponds to the abstraction of a hydrogen atom from AA by Tyr385* radical and formation of the E13 enzyme state containing AA* radical in the COX-site, . This AA* radical then reacts with two oxygen molecules and the intermediate product, PGG2* radical, is produced (reaction 3). The final step of the COX1 cycle in our model is the irreversible reaction 4, which describes the process of E5 state regeneration with Tyr385* radical, accompanied by the release of the intermediate product, PGG2 from the COX-site . Th. Th29]. e enzyme  and SimBin silico screening of the inhibition actions of different NSAIDs on PGHS-1 in in vitro experimental conditions. To simulate the in silico experiments on the NSAID interaction with PGHS-1 under in vivo conditions , we have upgraded our model in such a way that allows taking into account the synthesis and degradation of PGHS-1 in the cells , while in in vivo experiments with intact cells, the type of intracellular reducing cosubstrate and its level are unknown. Also, the type and the concentration of the reducing substrate, RC, in 50, depending on the experimental settings. In particular, it is known from the enzymatic theory that the dependence of the competitive inhibitor IC50 on the substrate concentration, S, may be written as:mK—Michaelis constant for the substrate, dK—the dissociation constant of inhibitor. Equation (1) shows that less competition of inhibitor with substrate at low substrate concentration (AA) causes the NSAID IC50 to decrease. Thus, the increase of NSAID effect in in vivo conditions may be expected. It has been shown that relation (1) holds in the wide range of the substrate concentrations for PGHS-1 [50 on AA concentration (1) is complicated by the complex kinetics of PGHS-1, particularly due to the existence of activation threshold in the range of low AA concentration [The factors mentioned above may change the value of NSAID’s ICr PGHS-1 . Howeverntration . Furthermore, reducing cosubstrate exerts a complex influence on the enzyme activity. Cosubstrate has been established to be an activator at low concentration and a suppressor of PGHS-1 activity at high concentrations of cosubstrate . in silico screening of oxygen consumption of PGHS-1 over a wide range of substrate and cosubstrate concentrations. To study the dependence of PGHS-1 activity on AA and RC concentrations in detail, we carried out an The obtained results show that the activation threshold of PGHS-1 depends not only on the internal properties of the enzyme , but alson,aspirink =10−5 μM−1 s−1[2 to PGH2 .According to the simulation of the dose dependence for aspirin shown in ls 50 μM . The expM–200 μM ,14. The 50, measured in vivo experiments, is much lower (1–3 μM), than the IC50 obtained in in vitro assays. To analyse this discrepancy, we carried out the in silico experiments, simulating the experiments on aspirin effect on platelets. Generally in such experiments, platelets are treated with the different concentrations of aspirin during the preincubation period of 25–30 minutes, and then the production of TxA2 is stimulated by calcium ionophore. The resulting concentration of TxB2 is then measured in 10–15 minutes [Extensive experimental studies of aspirin effect on intact cells  ,13,14,17 minutes . in silico experiment under in vivo conditions, we adjusted our method to the simulation of PGHS-1 catalysis in the platelet environment. The following assumptions were used in the calculation: (1) There is no de novo synthesis of PGHS-1 in platelets (synk = 0), but platelets contain a PGHS-1 depot. We estimated this initial concentration of PGHS-1 equal to 2.5 μM [2, produced after platelet stimulation [degk, was set as 10−5 s−1 based on the experimental estimation of PGHS half-life [in vitro experiments, as a cosubstrate.In the o 2.5 μM , based omulation . (2) PGHalf-life . (3) Int2 production. Thus we assumed that PGHS-1 functions in platelets at AA, RC concentrations close to its activation threshold , as compared to the IC50 estimate, derived from the in vitro one (100 μM). An analysis of the results obtained in in vitro and in vivo conditions allowed us to draw a conclusion on the possible mechanism, underpinning the stronger inhibitory effect of aspirin in vivo.As can be seen from the comparison of two theoretically derived dose dependencies shown in in silico experiment showed that this phenomenon originates from some complex enzyme dynamics, observed in the AA-RC concentration range close to the activation threshold of PGHS-1. In this range the interaction between catalytic activities of acetylated and non-acetylated (intact) forms of the enzyme contributes significantly to the inhibition of PGHS-1 (see scheme in 2 from the system (see 2[2 by the acetylated enzyme. Note that a kinetic interaction of acetylated and non-acetylated PGHS-1 results in a more pronounced effect on the enzyme inhibition at the concentrations of AA and RC close to the activation threshold (in vivo conditions), rather than far from one (in vitro conditions).The stem see . In thisem  [We applied the developed method to  PGHS-1) ,18, and  PGHS-1) . in silico the in vitro experiments on the inhibitory actions of celecoxib and ibuprofen on purified PGHS-1 [on,celecoxk = 1.6 μM−1 s−1, d,celecoxK = 0.2 μM and ibuprofen: on,ibuprofenk = 7 μM−1 s−1, d,ibuprofenK = 0.1 μM. Both the theoretical and experimental [50 of celecoxib and ibuprofen are in the range of 30 μM. The experimental values of the IC50 of ibuprofen lay in the wide range of the concentrations, 3.3 μM–100 μM [To estimate the affinities of these drugs to PGHS-1, we reproduced d PGHS-1 , and obtM–100 μM ,48,49,50in vitro conditions to the in vivo ones. The in silico screening of the drug action on platelet PGHS-1 was carried out at the concentrations of RC and AA near the activation threshold of the enzyme . According to this dependence, the fall in the AA concentration leads to the decrease in the IC50 value. Note that this mechanism, caused by the drug and AA competition, does not show up in the case of in vitro and in vivo experiments with platelets treated by slow irreversible inhibitor, aspirin. This effect is due to the fact that enzyme binds with aspirin in the absence of competitive substrate at the preincubation stage.The second reason for the decrease in IC50 (1 μM) is in agreement with the experimental values [50 are most likely to be explained by the variations in the experimental conditions used in different studies. For example, some experiments were performed on washed platelets [50 value [The obtained value of the celecoxib ICl values ,11,18, wl values ,11,18. Tlatelets . The metin vivo condition than in vitro one. The obtained value of the ibuprofen IC50 is 2 μM, which is in agreement with the experimental data, measured in the different experimental conditions: 7.6 μM (WBA) [The results of our calculations also showed that ibuprofen produces a stronger inhibitory effect on PGHS-1 μM (WBA) , 1.4 μM–μM (WBA) , 5 μM (bμM (WBA) . in silico screening of celecoxib and ibuprofen actions on PGHS-1 showed that these two drugs with different selectivity to PGHS-1 possess approximately equal abilities to inhibit PGHS-1 in platelets and in in vitro conditions. Below we continue to compare these NSAIDs by using in silico experiments in the different experimental conditions.The in vitro and in vivo experiments, as well as clinical studies, demonstrate that some nonselective NSAIDs  [The problem of NSAID co-administration is being actively discussed in literature in the context of uncertainty of the resulting therapeutic and side effects which arise from such combinations ,26,27. Baproxen) ,26 and Paproxen)  can undeaproxen) . But, thin silico screening of the resulting effect of two NSAIDs in combination on PGHS-1 in vivo and in vitro conditions. In this section, we demonstrate the application of our method to in silico study of the combined effects of aspirin and ibuprofen, as well as aspirin and celecoxib. To elucidate the mechanism of the interference of NSAIDs, we adapted our method to the in silico experiments we reproduced the conditions of the experiments on a combined action of aspirin with celecoxib, and aspirin with ibuprofen on PGHS-1 in vitro conditions and platelets [p-phenylenediamine), and the rate of cosubstrate oxidation was monitored spectrophotometrically [in silico experiment we used kinetic parameters of aspirin, ibuprofen, and celecoxib identified previously  faster than aspirin , and therefore tends to replace aspirin molecule in the COX-site at the high concentration of ibuprofen (celecoxib). It leads to saving some part of PGHS-1 from irreversible acetylation by aspirin. The substitution of aspirin by ibuprofen (celecoxib) starts when the drug concentration is in the range of the dissociation constants of ibuprofen, d,ibuprofenK = 0.1 μM or celecoxib, d,celecoxK = 0.2 μM  and arachidonic acid—all competing for the same COX binding site of the enzyme. These three small molecules differ by their kinetic properties—the rate of binding and the affinity to the COX-site of the enzyme, which results in an antagonistic effect between these species in the wide range of their concentrations. As has been established experimentally  and theo2 μM see . The sim= 0.1 μM ). Consid= 0.1 μM , AA inte50 (≅30 μM). The rise in the inhibition dose dependence curve observed in the range of 10 μM–100 μM of ibuprofen (celecoxib) concentrations is determined by the inhibition action of ibuprofen (celecoxib) in the region of its ICin vitro and in vivo experimental conditions, we carried out a computational simulation of the combined action of aspirin and ibuprofen (celecoxib) using the experimental conditions of the work [2 produced was measured after 10 min of the cyclooxygenase reaction. In the in silico experiment the intracellular concentration of AA and RC were chosen near the activation threshold of PGHS-1 (see discussion in The suppression effect of ibuprofen (celecoxib) on the aspirin-mediated inhibition of PGHS-1 has been confirmed in experiments on platelets  see exp. To analthe work . In thisin vivo case as well. However, there is the noticeable difference between these two cases. There is no restoration of the initial inhibition level in the range of ibuprofen (celecoxib) concentrations higher than 10 μM. The activation of platelets at these drug concentrations is determined by complete removal of ibuprofen (celecoxib) from intercellular environment of platelets [The theoretical dose dependencies for ibuprofen and celecoxib in the presence of 100 μM aspirin are shown in latelets . in silico experiment under the conditions of partial removal of celecoxib after the preincubation stage. The resulting dose dependence, given in in vitro case  is less than in in vitro case (30 μM) and close to the minimum of the dose dependence curve (at 1 μM). In this case the dip in the dose dependence curve is not formed. Note that the same effect of the disappearance of the blocking effect was observed in the case of the combination of aspirin and ibuprofen (data not shown). Thus, the in silico experiments showed that blocking effects of ibuprofen and celecoxib on the aspirin-mediated inhibition of platelet PGHS-1 depend significantly on the drug concentration in the intercellular environment. So the further in silico study of the conditions, at which NSAIDs block aspirin inhibitory effect on PGHS-1 in platelets, needs the consideration of pharmacokinetic data.Under in silico experiments on NSAID action on PGHS-1 was shown to be a promising tool to be applied to the following problems of in silico pharmacology:In silico screening of inhibition effects of different types of NSAIDs on PGHS-1 in various environmental conditions of the enzyme;in vitro assays, platelets, endothelia and other cells;Dissection of the key factors, which determine the variability of PGHS-1 response to NSAID action in In silico comparison of the different NSAIDs and the establishment of bioequivalence of the various drugs in the different experimental conditions;in vitro experimental conditions to in vivo settings;Translation of the results on NSAIDs action on PGHS-1, obtained in In silico study of the drug interference, underlying the combined action of two NSAIDs on PGHS-1 to predict the potential risks and benefits of NSAID combination therapy.The developed method of the in silico screening of PGHS-1 inhibition by NSAIDs in the intracellular environments corresponding to the inflammation progression in different cells may be a useful technique for predicting the anti-inflammatory and adverse effects of NSAIDs and their combinations in the changeable intracellular conditions. The proposed method of the


Estrogen Receptor alpha (ERα)-positive breast cancer patients receive endocrine therapy, often in the form of tamoxifen. However, resistance to tamoxifen is frequently observed. A signalling cascade that leads to tamoxifen resistance is dictated by activation of the Protein Kinase A (PKA) pathway, which leads to phosphorylation of ERα on Serine 305 and receptor activation, following tamoxifen binding. Thus far, it remains elusive what protein complexes enable the PKA-ERα interaction resulting in ERα Serine 305 phosphorylation.In vitro analyses were performed using MCF7 breast cancer cells, which included immunoprecipitations and Fluorescence Resonance Energy Transfer (FRET) analyses to illustrate ERα complex formation. siRNA mediated knockdown experiments were performed to assess effects on ERαSerine 305 phosphorylation status, ERα/PKA interactions and downstream responsive gene activity.We performed immunohistochemistry to detect ERαSerine 305 phosphorylation in a cohort of breast cancer patients who received tamoxifen treatment in the metastatic setting. From the same tumor specimens, Agilent 44 K gene expression analyses were performed and integrated with clinicopathological data and survival information. Stratifying breast tumors on ERα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13. AKAP13 mRNA expression levels correlate with poor outcome in patients who received tamoxifen treatment in the metastatic setting. In addition, AKAP13 mRNA levels correlate with ERαSerine 305 phosphorylation in breast tumor samples, suggesting a functional connection between these two events. In a luminal breast cancer cell line, AKAP13 was found to interact with ERα as well as with a regulatory subunit of PKA. Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERα and abrogated PKA-driven tamoxifen resistance, illustrating that AKAP13 is an essential protein in this process.We show that the PKA-anchoring protein AKAP13 is essential for the phosphorylation of ERαS305, which leads to tamoxifen resistance both in cell lines and tamoxifen-treated breast cancer patients.The online version of this article (doi:10.1186/s12885-015-1591-4) contains supplementary material, which is available to authorized users. Breast cancer is the most frequently diagnosed malignancy in women. Since 75 % of all breast tumors express Estrogen Receptor alpha (ERα), tumor growth is considered to be dependent on the activity of this hormone-induced transcription factor. Thereby, treatment is focused on inhibiting the function of ERα. One of the most frequently prescribed drugs in endocrine treatment is tamoxifen. Tamoxifen competes with ERα’s natural ligand estradiol for binding to the ligand-binding pocket of the receptor. Tamoxifen forces the receptor in an alternative conformation as compared to estradiol, thereby preventing recruitment of coregulators to the complex, which are essential for ERα-driven transcription . Althougin vitro that PKA-RIα knockdown enhances breast cancer cell proliferation in the presence of tamoxifen. In addition, we showed in the same study that the major site PKA-responsive phosphorylation site on ERα is a serine residue found at position 305. This phosphorylation leads to a conformational arrest within the receptor and results in an agonistic response of the otherwise inhibitory compound tamoxifen. In this altered conformation ERα re-orientates its C-terminus towards its coactivators, which prevents a dissociation of RNA Polymerase II from the complex that is normally observed in tamoxifen treated cells, thereby resulting in transcriptional activation in the presence of tamoxifen [An alternative mechanism of tamoxifen resistance is mediated by activation of the Protein Kinase A (PKA) pathway . Decreasamoxifen . PKA-stiamoxifen  This potamoxifen .in vitro data, in breast cancer patients treated with tamoxifen for metastatic disease, the phosphorylation status of ERαS305 was found to be indicative for poor outcome [In addition to  outcome . Moreove outcome , 15 and  outcome . HoweverPKA is a multi-protein complex. The inactivated PKA complex is composed of a catalytic subunit (PKAcat) that associates, in an inactive state, to its regulatory subunits (PKA-RI and RII). After the regulatory subunits bind cAMP, the catalytic subunit can dissociate from the complex to phosphorylate its substrates. There are many known PKA targets, and in order to achieve substrate-specificity, PKA activity needs to be locally confined. Localized PKA activation can occur in multiple ways. Intracellularly, cAMP levels are unevenly and dynamically distributed, , 18 whicIn vitro experiments could illustrate that AKAP13 interacts with ERα as well as the regulatory subunit of PKA, and that AKAP13 expression is essential for PKA-induced ERαS305 phosphorylation. In summary, we demonstrate here that the PKA-mediated phosphorylation of ERα at Serine 305, which leads to tamoxifen resistance, requires the PKA-anchoring protein AKAP13.Here, we studied the enrichment of PKA-associated molecular pathways in a cohort of breast cancer patients, which received tamoxifen in a metastatic setting. Stratifying tumors on ERαS305 phosphorylation status resulted in the identification of a molecular pathway involving AKAP13, suggesting a functional link between AKAP13 levels and PKA-induced ERα phosphorylation. AKAP13 expression levels correlated with a poor outcome after tamoxifen treatment in breast cancer patients and correlated with ERαS305 phosphorylation status. MCF-7 cells were cultured in DMEM medium, supplemented with 8 % fetal-bovine serum and standard antibiotics. The following antibodies were used: AKAP13 (Bethyl A301-404A-1), ERα (Santa Cruz sc-543), ERαS305P , PKA RII (Abcam ab57414), PKA catalytic-α , beta-actin  and AKAP95 . 3 days prior to cell biological analyses, cells were switched to phenol-red free DMEM, supplemented with 5 % charcoal-treated serum. For siRNA targeting of AKAP13 and AKAP95, a smartpool of four unique siRNAs was applied (Thermo Scientific Dharmacon) using the manufacturers’ protocols. Cells were subsequently cultured for 72 h on hormone-deprived phenol-red free DMEM. The expression vector for ERα-CFP was previously described [4 cells/ well in phenol-red free DMEM, supplemented with 5 % charcoal-treated serum. Compounds were introduced the following day and cells were allowed to grow in the presence of the compounds for 2 weeks. The compounds tested were 17β-estradiol (10−8 M), 4-OH-tamoxifen (10−7 M) and Fulvestrant (10−7 M), while incubation in the presence of vehicle alone (DMSO) was used as a control. After 2 weeks cells were fixed with methanol and stained by a 0.2 % crystal violet solution. Finally, the dye was solubilized in a weak acetic acid solution and the optical density measured at 590 nm using the Infinite® 200 reader (TECAN). Alternatively, growth was assessed by MTT using the manufacturers recomendations (Sima-Aldrich).MCF-7 cells were switched into phenol-red free DMEM supplemented with 5 % charcoal-treated serum. After 24 h, cells were transfected with siAKAP13, siAKAP95 or siCntrl. After 2 days the cells were seeded in 48 well plates at a density of 10For immunohistochemistry, tissue microarrays (TMAs) were used from formalin fixed-paraffin-embedded tumor tissue. Antigen retrieval was performed using citrate buffer citrate buffer . Citrate buffer was pre-heated and slides were subsequently added for 15 min (microwave 300 W). The ERαS305P antibody staining was performed overnight in 1:20 dilution (Millipore # 124-9-4). No lower cut-off for positivity was applied, and any ERαS305 positive signal in the entire slide was scored as positive. This mode of scoring was consistent as previously described for this antibody , 35.MCF-7 cells were grown in DMEM until ~40 % confluency was achieved. Cells were subsequently hormone depleted by culturing in heat inactivated charcoal-treated serum-containing medium for 3–4 days to block all ERα activity. Prior to immunoprecipitation, cells were washed and lysed according to standard protocols. Immunoprecipitation was performed overnight with anti PKA RII, AKAP13, ERα or negative control anti FLAG-M2 (Sigma). Thereafter, the beads were washed and prepared for Western blot analysis.For quantitative RT-PCR analyses, cells were seeded in 12 well plates and hormone-deprived for 3 days. After six hours of hormonal treatment, RNA was isolated using Trizol (Life Technologies) using the manufacturers protocols. cDNA was generated using SuperScript III (Life Technologies) using the manufacturers protocols. Primers used for QPCR were for XBP1 (GGGAAGGGCATTTGAAGAAC (FWD); ATGGATTCTGGCGGTATTGA (REV)) and TFF1 (ATCGACGTCCCTCCAGAAGA (FWD); TGGGACTAATCACCGTGCTG (REV)). As housekeeping gene, GAPDH (GCCATCAATGACCCCTTCAT (FWD); TGACAAGCTTCCCGTTCTCA (REV) was used.2. FLIM experiments were performed using a LEICA DM-IRE2 microscope equipped with a Lambert Instruments frequency domain lifetime attachment. CFP was excited at 430 nm. Emission was collected at 450–490 nm using a CCD camera. FLIM measurements were performed using U2Os cells, transfected with ERα-CFP, PKA-cat-YFP and a non-tagged PKA-regulatory subunit. Cells were co-transfected with siAKAP13 or siControl. Calculated CFP lifetimes were referenced to Rhodamine-G6 which was set at 4.11 nsec, and internally calibrated using cocultured CFP containing MelJuso cells for which the lifetime was set to 2.7 nsec, as we applied before [D) was calculated as ED = 1- (lifetime cell of interest/ lifetime reference cell). Pairwise analysis was performed for each cell before and after treatment with 10 μM forskolin for 1 h, where ED under CTS conditions was set to 1 for each experiment.FRET was performed using Fluorescence Lifetime Imaging Microscopy (FLIM) as we applied before . Cells wd before . Donor Fhttp://www.fmwv.nl) and has been approved by the local medical ethics committee of the Netherlands Cancer Institute. The use of anonymous or coded left over material for scientific purposes is part of the standard treatment agreement with patients and therefore informed consent was not required according to Dutch law [The patient series used in this paper has been previously described in detail . This stutch law .For the analysis of pathway enrichment in ERαS305P positive breast cancer patients we used a series of breast cancer patients who were treated with tamoxifen for metastatic disease as was previously described , 35. In P-value was computed for each gene in a pathway. Then the set of P-values for a pathway was summarized by the LS summary statistics. For a set of N genes, the LS summary statistic (LS = ∑Ni=1 (−log(pi))/N) was defined as the mean negative natural logarithm of the P-values of the appropriate single gene univariate test. Second, we related the pathways to PKA using the Cancer Genome Anatomy Project (http://cgap.nci.nih.gov/Genes/GeneFinder) and 27 pathways out of the 302 were found to be related to PKA. Next, we tested whether the list of significant pathways as defined by the LS statistic (P < 0.05) was enriched for PKA-related pathways using Fisher’s Exact test.The analysis for pathway enrichment in ERαS305P positive patients and Agilent 44K data was previously described . Data anU test and the Chi square test for trend. Data were analyzed using SPSS 15.0.Time to tumour progression (TTP) was considered the primary endpoint measured from the start of tamoxifen administration and until treatment was ended because of tumour progression. TTP was estimated according to the Kaplan–Meier method for four different AKAP13 probes, segmenting the continuous variable in two groups (low and high) with an equal number of events. In addition we tested the four different AKAP13 probes for trend, using the significance of the coefficient for the continuous AKAP13 variable. For the probe with the most significant test for trend, we compared TTP in the NKI cohort for two subgroups (low and high) by uni- and multi-variate Cox proportional hazards regression. Clinico-pathological characteristics (only available for NKI cohort) according to AKAP13 levels were compared using Fisher’s exact, Mann-Witney Serine 305 phosphorylation on ERα was found to be a predictive marker for tamoxifen resistance in breast cancer patients , 15. To in vitro whether this enrichment also implies a causal role for either of these two proteins in PKA-mediated ERα Serine 305 phosphorylation. siRNA-mediated knockdown of AKAP13 and AKAP95 was performed in MCF-7 breast cancer cells . We then compared clinico-pathological characteristics for two subgroups (low and high) of this probe, Table p = 0.033) and there was a trend towards negative progesterone receptor status (p = 0.071). Using a univariate Cox proportional hazard model we found a hazard ratio of 2.10 for patients with high AKAP13 levels (p = 0.007). Other clinico-pathological characteristics that were significant in the univariate analysis were ERα level  and PR status (HR 0.45 p = 0.004)   was estimated according to the Kaplan–Meier method for AKAP13 expression, segmenting the continuous variable in two groups (low and high) with equal numbers of patients Fig. . Four diin vitro , and are considered to grow dependent on the activity of this transcription factor. About 50 % of breast cancer cases without distant metastatic disease can be cured by surgery alone  and endoin vitro, siRNA targeting AKAP13 did prevent ERαS305 phosphorylation. These data highlight the necessity for in vitro verification of findings based on correlations in patient subpopulations before drawing conclusions that suggest any causality.Kinases play a central role in endocrine resistance, with many (receptor tyrosine) kinases being differentially expressed or differentially activated in endocrine treatment resistant breast cancer. Examples of these include EGFR , ErbB2 , IGF-1R As a model for PKA-mediated ERαS305P phosphorylation, leading to tamoxifen resistance, we propose the following order of events  has been described to correlate with high-risk familial breast cancer , 34. In Kinases are promising drug targets in cancer treatment. Still, when subcellular localization and activity are key determining factors rather than global activation status of a kinase, the pharmacological inhibition of the total kinase pool could potentially result in a significantly high level of toxicity. Inhibiting the specific interactions of kinases with their localization-confining factors may provide a highly specific inhibition of a kinase subset, while the total pool of such a kinase remains unaffected. With respect to the current study, the interface between ERα and AKAP13 could be a suitable drugable interaction, through which this form of tamoxifen resistance could potentially be inhibited.Breast cancer stratification on ERαS305 phosphorylation status led to the identification of AKAP13 as a potential mediator for this phosphorylation event on ERα. AKAP13 expression levels correlate with response to tamoxifen treatment in the metastatic setting. Tumors with high AKAP13 mRNA levels are enriched for ERαS305P positivity on IHC. Using cell lines, we have shown that AKAP13 interacts both with ERα and PKA-RII, which enables PKA-induced ERα phosphorylation and tamoxifen-driven gene expression in a tamoxifen-resistant cell line with elevated PKA activity. With this, AKAP13 plays a key function in enabling PKA-induced ERα phosphorylation and is causally involved in ERαS305P-mediated tamoxifen resistance.


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  and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1βMYPT1 phosphatase.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 SCF MYPT1 and SCFβTrCP signalling components. It demonstrates NUAK1 inhibitors suppress cell proliferation and PLK1.The present study provides insights into the biological regulation of the NUAK isoforms and highlights the remarkable interplay that exists between Polo kinase, NUAK1, PP1β All DNA constructs were verified by DNA sequencing, which was performed by the Sequencing Service , using DYEnamic ET terminator chemistry  on Applied Biosystems automated DNA sequencers. Cell proliferation assay was carried out using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit as described previously  mutant, were markedly reduced (To investigate whether the expression of NUAK1 was controlled by the SCF reduced A.http://www.biochemj.org/bj/461/bj4610233add.htm).Treating cells with calyculin A to enhance NUAK1 phosphorylation induced significant degradation of endogenous NUAK1 within a 2–4 h time frame B. This eTo obtain further evidence that βTrCP1 regulates NUAK1, we investigated how knockout of the βTrCP1 isoform in previously described MEF cells  impacted476 and Ser480, we investigated the effect of a variety of selective protein kinase inhibitors known to target kinases that phosphorylate βTrCP-binding phosphodegron motifs on other targets. These included inhibitors of IKKs (BI605906), GSK3β (CHIR99021), polo-kinases (BI2536 and GSK461364), CK1 (D4476), aurora kinase (VX680) and CDKs (roscovitine). We also tested a number of selective inhibitors of major protein kinase signalling systems namely ATM , DNA-PK , ATR , mTOR , PI3K  and Akt (MK2206). This analysis strikingly revealed that two structurally unrelated inhibitors of polo kinases , namely BI2536  mutant proliferated at nearly 2-fold-higher rates than U2OS cells expressing WT NUAK1 or control cells not overexpressing NUAK1 C. We alsMYPT1 binds to and inhibits PLK1 by dephosphorylating the T-loop residue (Thr210) [MYPT1 to bind to and dephosphorylate and hence inactivate PLK1 is dependent upon phosphorylation of MYPT1 at Ser473 by the CDK1, which creates a binding site for the PLK1 Polo-box domains [473 lies adjacent to the NUAK1 phosphorylation site (Ser472) that controls 14-3-3 binding [445 and Ser472  . The abi domains . As Ser4 binding , we deci binding . Immunobd Ser472 . Strikin protein .βTrCP E3 ubiquitin ligase complex. Our findings suggest that PLK by phosphorylating NUAK1 at Ser476 and Ser480 triggers interaction with the SCFβTrCP complex resulting in ubiquitylation and degradation of NUAK1. This model is supported by NUAK1 expression being increased following inhibition of PLK (with BI2536 or GSK461364), mutation of Ser476/Ser480 or inhibition of the SCF complex (with MLN4924). We also demonstrate that in phases of the cell cycle where PLK1 is most active (G2–M), NUAK1 levels are low and vice versa when PLK1 is less active (late G1 to S), NUAK1 expression increases. Ser476 and Ser480 are highly conserved and located within the C-terminal domain of NUAK isoforms. To our knowledge Ser476/Ser480 have not been previously been documented (http://www.phosphosite.org/).Our data demonstrate that NUAK isoforms are novel substrates for PLK1 and SCFβTrCP binding thereby leading to ubiquitylation and degradation of the target. These include the Bora-activating subunit of the aurora A kinase [SGYYS motif encompassing Ser476 and Ser480 in NUAK1 as well as the equivalent residues in NUAK2 isoforms  on NUAK1 and/or the βTrCP-binding residues (Ser476 to Ser480)  residue and thereby PLK1 inactivation [473 [472, lies adjacent to Ser473 that controls PLK1 binding to MYPT1. Phosphorylation of Ser472 triggers 14-3-3 isoforms binding to MYPT1 [210 phosphorylation of PLK1. This is also accompanied by an electrophoretic band shift, suggesting that stoichiometry of phosphorylation is significant  . PLK1 hativation ,51. Bindion [473 . Interesnificant . This ph mitosis  and cellMYPT1, SCFβTrCP, PLK and CDK components (MYPT1 complex. However, once PLK1 activity is increased, this should result in phosphorylation of Ser476 and Ser480 of NUAK1, at least once CDKs are activated. This would in turn promote SCFβTrCP binding and hence result in ubiquitylation and degradation of NUAK1. Reduced NUAK1 level and activity will result in increased activity of PP1βMYPT1 complex,, which will then inhibit PLK1 by inducing dephosphorylation of the T-loop of PLK1.Our data suggest that there is a remarkable interplay between NUAK1, PP1βmponents . ConditiMYPT1 and PLK1 [MYPT1 and PLK1, the authors of that study observed that shRNA-mediated knockdown of LKB1, that would be expected to inhibit NUAK1 activity, promoted the association of MYPT1 and PLK1 [A recent study reports that the LKB1 pathway plays a role in regulating the centrosome via NUAK1, PP1βand PLK1 . Consistand PLK1 . Howeverand PLK1 . This co476 and Ser480 are ablated also significantly accelerated cell proliferation (βTrCP, that orchestrates ubiquitylation and degradation of key cell cycle co-ordinators [MYPT1 has also been implicated in mediating important roles in the cell cycle, by dephosphorylating PLK1 as well as other proteins predicted to regulate kinetochore, centrosomes and central spindle [2–M-phase of the cell cycle, we believe that NUAK1 is playing a vital role in the S-phase and hence inhibition of NUAK1 leads to defects in S-phase which progressively leads to mitotic defects as well. In future work it would fascinating to explore in more depth the interplay between NUAKs, PLK1, PP1βMYPT1 and SCFβTrCP to better define the role that this system plays in regulating cell proliferation. Both BI2536 and GSK461364 potently inhibit PLK2 and PLK3 as well [We also found that treatment of cells with structurally diverse highly selective NUAK1 inhibitors (WZ4003 and HTH-01-015 [feration C. The fidinators , further spindle ,51. Alth as well , so alth


Many successful attempts to increase pullout strength of pedicle screws in osteoporotic bone have been accompanied with an increased risk of catastrophic damage to the patient. To avoid this, a single-armed expansive pedicle screw was designed to increase fixation strength while controlling postfailure damage away from the nerves surrounding the pedicle. The screw was then subsequently tested in two severely osteoporotic models: one representing trabecular bone (with and without the presence of polymethylmethacrylate) and the other representing a combination of trabecular and cortical bone. Maximum pullout strength, stiffness, energy to failure, energy to removal, and size of the resulting block damage were statistically compared among conditions. While expandable pedicle screws produced maximum pullout forces less than or comparable to standard screws, they required a higher amount of energy to be fully removed from both models. Furthermore, damage to the cortical layer in the composite test blocks was smaller in all measured directions for tests involving expandable pedicle screws than those involving standard pedicle screws. This indicates that while initial fixation may not differ in the presence of cortical bone, the expandable pedicle screw offers an increased level of postfailure stability and safety to patients awaiting revision surgery. Osteoporosis is a global public health problem affecting over 200 million people worldwide, with 44 million affected in the United States . CliniciMost current procedures in spinal fusion restrict mobility in one or multiple levels of the spine so that, over time, arthrodesis can occur. The current clinical “gold standard” of spinal fusion is the bilateral pedicle screw system . HoweverTo overcome this issue, some researchers have considered a variety of alterations to screw design or insertion technique to increase the amount of fixation in low density bone. However, many of the more successful techniques also involve an increased level of risk to the patient. For instance, increasing the diameter of a pedicle screw has been shown to increase the purchase between thread and bone –7, but pInvestigations often use pullout testing to determine the fixation efficacy of pedicle screws in the human vertebra regardless of bone quality. They are often performed in laboratory conditions on either cadaveric , 14, 19 Considering the dangers associated with many of the above techniques, the question arose if it were possible to design a pedicle screw that would increase pullout strength when instrumented in osteoporotic individuals while avoiding catastrophic damage to the patient should failure occur. Therefore, the aim of this investigation was to test a concept of pedicle screw design that limited radial expansion to a single direction and obtain preliminary data on its stability and potential safety through axial pullout tests in polyurethane test blocks. It was believed that the addition of a single expandable arm would not only provide the expandable pedicle screws superior pullout strength over the standard screws but also result in similar measurements of synthetic bone loss between the two screw types in all directions surrounding the screw except for the direction of the expandable arm, which is expected to remove a greater amount of polyurethane foam exclusively in that direction. Clinically, the ability to position the expandable arm in the superolateral direction of the pedicle should theoretically control bone failure in a way that avoids potential catastrophic damage to the nerves surrounding the pedicle. in vivo, the arm could be positioned in the superolateral direction to the pedicle. This way, should bone fracture occur with this screw, it was proposed that any additional damage to the vertebral body over what would normally be seen in a standard pedicle screw would be located in an area away from potential harm of the surrounding nerves.Twelve (12) 6.5 × 45 mm Ti-6Al-4V standard pedicle screws were used in this investigation . The tit3, a density similar to that of severely osteoporotic trabecular bone. The other block, referred to as composite block, was designed to simulate the interaction of the expandable arm as it makes contact with cortical bone. This block consisted of sections of varying densities to simulate both the trabecular and cortical bone comprising severely osteoporotic vertebral bodies. The portion of the block representing trabecular bone measured 20.25 mm × 30 mm × 30 mm and had a density of 0.08 g/cm3. The sections of the block representing the cortical bone were 2.25 ± 0.25 mm thick and had a density of 0.64 g/cm3. This higher density portion was layered along the top and one of the sides of the trabecular section. The thinner design of the composite block in comparison to the trabecular is to allow the arm of the pedicle screw to open within the proximity of the cortical wall while permitting the screw to be in the center of the testing machine.Testing blocks were obtained from Sawbones . Two sets of polyurethane blocks of different sizes and compositions were used in the current study . One, reThe PMMA used in this study was provided by DFINE, Inc. , and was composed of a powder  and liquid monomer . Approximately 2.0 mL of the cement was injected into the test block prior to screw insertion for each PMMA test. The cement had a working time of thirty minutes. All screws were implanted within this window and then the cement was allowed to fully harden for over 12 hours prior to testing.The pedicle screws were inserted into the test blocks by placing a specially designed cap over the blocks to locate proper placement of the pilot hole and to increase confidence that the screw was inserted perpendicular to the top surface. Markings on the screws indicated not only the proper depth to which the screws were to be inserted (45 mm in the trabecular blocks and 30 mm in the shorter composite blocks) but also the location that the expandable arm was facing inside the foam. All screws were driven up until the marking reached the surface of the test block while ensuring at that point that the expandable arm, if applicable, was positioned in its desired location . The arms on the expandable screws were expanded after insertion and proper expansion of the arm was confirmed using an OEC 9400 C-arm .n = 6 for each): (1) standard pedicle screws in trabecular block, (2) expandable pedicle screws in trabecular block, (3) PMMA augmented standard pedicle screws in trabecular block, (4) PMMA augmented expandable pedicle screws in trabecular block, (5) standard pedicle screws in composite block, and (6) expandable pedicle screws in composite block. Test order was randomized among all conditions, except for the ones involving bone cement augmentation (conditions 5 and 6), which were tested last since PMMA injection was considered to create irreversible alterations to the screws.Pullout testing was performed in six conditions . The hydraulic clamp, attached to the base of the machine, kept the pedicle screw stationary during pullout testing. The fixation frame, which was attached to the biaxial load cell, ensured that the test blocks were held stationary relative to the load cell and did not wobble or become displaced in any other manner. The weight of the fixation frame was taken into consideration by zeroing the output values of the load cell prior to testing. Axial displacement occurred at a rate of 5 mm/min and continued until the pedicle screw was completely removed from the test block. At that point, the damage to the test block was measured at each desired level using a set of digital calipers (±0.03 mm) centered at the original location of screw placement and in the directions parallel to the sides of the block, as illustrated in Mechanical parameters recorded during pullout testing included pullout force, stiffness, energy to failure, and energy to removal. Pullout force (N) was defined as the maximum axial load achieved during the entirety of the test. Stiffness (N/mm) was defined as the approximate slope on the load displacement curve leading up to the point of screw pullout. Energy to failure (N∗mm) was defined as the area under the load displacement curve up until pullout was reached. Energy to removal (N∗mm) was defined as the area under the entirety of the load displacement curve.t-tests were performed for comparing all values between the standard and expandable pedicle screw conditions. For comparisons between PMMA augmented and nonaugmented screws of the same type, a paired t-test was used to determine differences between each of the mechanical parameters while a Wilcoxon signed rank test was used to determine differences between the measurements of damage. A significance level of 0.05 was used for all statistical comparisons. The power for all statistically significant comparisons observed was at or above 80%, unless otherwise noted .Statistical analysis was performed using SAS 9.2 . Using a Shapiro-Wilk test, normality was verified for all measurements and mechanical parameters except for the damage size produced by PMMA augmented screws. Thus, Due to a malfunction of the C-arm during the testing procedure, six (6) of the blocks instrumented with expandable pedicle screws could not be imaged. Therefore, to determine if the arm was properly expanded, the photographs of all the expandable screws were reevaluated after the pullout testing was performed. If the arm exited the block fully extended, it was determined that the arm was open enough to “catch” the foam block as it was being pulled out and adequate expansion was confirmed. On the other hand, if the arm failed to be pulled open by the surrounding low density foam, it was determined that the arm was not initially opened to an appropriate angle and the results for that individual test were disregarded. There were three (3) such instances of this happening, in which case additional pullout tests in new testing blocks had to be performed.The mean and standard deviation of the values returned for each mechanical parameter can be found in P < 0.01) while showing no statistical differences in stiffness (P = 0.07) or energy to failure (P = 0.11) and an increase in energy to removal of 26.3% (P < 0.001). PMMA augmentation of the standard pedicle screws produced no noticeable differences in pullout (P = 0.98), stiffness (P = 0.06), or energy to failure (P = 0.43) when being compared to the nonaugmented standard pedicle screw. It did, however, increase the energy to removal of the standard screws by 97.3% (P < 0.001). PMMA augmented expandable pedicle screws tested against nonaugmented expandable screws demonstrated an increase in pullout strength of 12.3% , energy to failure of 33.4% (P = 0.01), and energy to removal of 94.6% (P = 0.001). PMMA augmentation, however, did not produce any statistical differences in stiffness in the expandable pedicle screws (P = 0.50).For tests performed in trabecular blocks, the expandable pedicle screws produced a mean pullout force 5.4% less than standard pedicle screws (P = 0.47), stiffness (P = 0.86), and energy to failure (P = 0.69). There was, however, a significant increase in the energy to screw removal as the expandable pedicle screws produced mean values 46.3% greater than standard pedicle screws (P = 0.001).In the composite blocks, the expandable pedicle screws produced no statistical differences compared to the standard pedicle screws with regard to pullout force , the addition of the expandable arm increased the distance of block damage by 47.4% (P < 0.01). PMMA augmentation of the standard pedicle screws increased block damage by 122% (P = 0.03) to 212% (P = 0.03) when compared to the nonaugmented standard pedicle screws. Similarly, augmentation of the expandable pedicle screws increased the amount of trabecular block damage by 58.7% in both tests' maximum damage recording (P = 0.03) and 104% in the direction of both tests' minimum amount of recorded damage (P = 0.03).While the expandable pedicle screw produced similar amounts of foam removal to the standard screw as a result of its outer threading , the expandable arms increased the distance of foam removal by 38.7% in that direction (P < 0.01). In the top cortical layer, the expandable pedicle screws created significantly less damage in all measured directions than the standard screws. The bottom side of this layer had foam removal averaging 23.7% less in the direction of arm expansion  and 27.4% less in the direction perpendicular to arm expansion (P = 0.01) while the top surface averaged 13.8% smaller in the direction of the arm  and 18.1% smaller in the perpendicular direction (P < 0.01).While the expandable pedicle screws produced a similar amount of low density foam removal as the standard pedicle screws due to the screw's threading . This shows the balance between safety and strength of this design.It is to be acknowledged that limitations to this study are present. Largely due to the high cost of pedicle screws, six standard screws and six expandable screws were used allowing for a maximum sample size of six for all test conditions. However, to enhance reproducibility of the results, homogeneous synthetic bone models were used in place of cadaveric human vertebra of varying bone mineral densities.An additional limitation lies in the composite block. While it was designed to serve as an analog emulating how the screw would interact with the surrounding cortical wall, it has not been verified via mechanical comparisons between it and cadaveric models of a similar density. Therefore, future studies with this model should be performed in conjunction with cadaveric specimens and a large enough sample size to sufficiently increase the power of the study.Final recommendations for future research can be made on the design of the expandable pedicle screw. In order to increase pullout strength, a wider arm or additional arms could be strategically incorporated to the screw in such a way that they maximize the surface area making contact with the cortical wall of the vertebral body while still expanding in specific locations  to represent the least risk of neurological damage in case of failure. Moreover, an arm-retraction mechanism should be incorporated to avoid damage to the pedicle in the event that the screw needs to be removed after implantation. This was not included in the current design as the purpose of the study was to test the feasibility of a new design concept rather than testing a screw about to be used clinically. Adding such a mechanism at this stage would have significantly increased production time past what is desired at such an early phase in the product's development.The single-armed expandable pedicle screw design could not outperform a standard pedicle screw in a pullout test performed in a synthetic model; however, the more gradual decrease in axial load following pullout observed with the expandable screw may potentially result in a higher level of stability as the patient waits for revision surgery in the case of instrumentation's failure. Furthermore, the expandable pedicle screw was successful in limiting damage to the desired location, offering a minimum risk of neural damage in the event of pullout. Damage to the cortical layer of the composite block revealed that the expandable pedicle screw may be as safe as, if not safer than, a standard pedicle screw instrumented in an osteoporotic vertebra.


Egg specific IgE levels are frequently used in combination with skin-prick tests to guide clinical decisions and to monitor egg allergy evolution in children. We compared both Immulite and ImmunoCAP egg specific IgE assays in egg allergic children, and found a linear correlation between both assays with a mean Immulite:ImmunoCAP ratio of 3. This is relevant information for clinicans wishing to estimate values from one assay to the other, as most literature has been published using the ImmunoCAP system. Egg allergy represents one of the most common food allergies encountered in pediatric practice, with an estimated prevalence of 0.5–2 % in infants and young children . Many egThe objective of this study was to directly compare Immulite and ImmunoCAP egg white-specific IgE assays and to determine whether their measurements can be applied equivalently and/or adapted to guide clinical management of egg allergic children.Briefly, 37 egg allergic patients between 2 and 13 years of age were enrolled at Sainte-Justine University Hospital Center  from July 2013 to January 2014. Patients with egg allergy had either a positive OFC or a history of at least one sign or symptom of allergy  occurring within 1 h of egg ingestion and persistent sensitization at time of evaluation confirmed by a positive egg white skin prick test (3 mm greater than control), and either ImmunoCAP specific IgE levels ≥0.35 kU/L or Immulite specific IgE levels ≥0.1 kU/L. The project was approved by the ethics committee of Sainte-Justine University Hospital Center.Patients’ serum was aliquoted into two separate samples and sent on dry ice for analysis at the laboratories of the University of Montreal Hospital Center (CHUM) and Sainte-Justine University Hospital Center, each using a different specific IgE assay system: ImmunoCAP Phadia 250 and Siemens DPC Immulite 2000.Descriptive analysis consisted of medians and range. Immulite and ImmunoCAP values were compared using Pearson’s correlation .The median age of patients was 6.5 years  and the median age at first reaction to eggs was 12 months . The age of worst reaction was a median of 5.5 years before testing. Eighteen patients (49 %) had a history of anaphylactic reactions to eggs and 10 (27 %) tolerated baked eggs, while most of the remainder had never ingested baked egg before. Median egg white skin prick test diameter at time of specific IgE measurement was 10 mm .In the whole cohort, Immulite median egg white-specific IgE levels was 24.80  kU/L compared to 6.45  kU/L for ImmunoCAP. In the subgroup tolerating baked eggs (n = 10), median egg white-specific IgE levels was 5.2  kU/L using Immulite and 3.17  kU/L using ImmunoCAP. When examining the subgroup of patients with anaphylactic reactions to eggs (n = 18), the median egg white-specific IgE levels using Immulite was 17.4  kU/L compared to 5.90  kU/L for ImmunoCAP.Thus, as previously suggested, Immulite and ImmunoCAP egg-specific IgE values could not be substituted . HoweverThese observations are in line with previous studies. Wang et al. found an Immulite:ImmunoCAP ratio of 3.7 for egg-white specific IgE in 50 atopic patients . AlthougIn conclusion, because of variability between Immulite and ImmunoCAP specific IgE assays, it is preferable to use a single assay to monitor the evolution of egg allergy and to assess the development of tolerance. This said, a linear correlation does exist between both assays, as has been observed in four independent cohorts including ours. Therefore in the absence of access to ImmunoCAP, a factor of 3–5 could be applied to egg-specific IgE published thresholds to guide clinical decisions. Although imperfect, this approach remains in our opinion preferable to withholding useful clinical information from patients and clinicians.


This report is the first to demonstrate that the direct inhibition of PAX3-FOXO1 phosphorylation reduces ARMS tumor phenotypes in vitro and that these phosphorylation events are present in primary human ARMS tumors and invading tumor cells. These results identify phosphorylation of PAX3-FOXO1, especially at Ser201, as a novel biological target that can be explored as a promising avenue for ARMS therapies.Patients with translocation-positive alveolar rhabdomyosarcoma (ARMS), an aggressive childhood tumor primarily characterized by the PAX3-FOXO1 oncogenic fusion protein, have a poor prognosis because of lack of therapies that specifically target ARMS tumors. This fact highlights the need for novel pharmaceutical interventions. Posttranslational modifications such as phosphorylation are becoming attractive biological targets for the development of such interventions. Along these lines, we demonstrated that PAX3-FOXO1 is phosphorylated at three specific sites and that its pattern of phosphorylation is altered relative to wild-type Pax3 throughout early myogenesis and in ARMS tumor cells. However, little work has been performed examining the effect of directly inhibiting phosphorylation at these sites on ARMS development. To address this gap in knowledge, we used small molecule inhibitors or mutational analysis to specifically inhibit phosphorylation of PAX3-FOXO1 to investigate how altering phosphorylation of the oncogenic fusion protein affects ARMS phenotypes. We found that inhibiting the phosphorylation of PAX3-FOXO1 at Ser201 significantly reduced migration, invasion and proliferation in two independent ARMS tumor cell lines. Further, we found that inhibition of phosphorylation at Ser205 also decreased proliferation and anchorage-independent growth. Consistent with these  ARMS, the more aggressive subtype, is primarily defined by the t translocation, which fuses the amino-terminal region of Pax3 to the carboxyl-terminal sequences of FOXO1.4 The resulting PAX3-FOXO1 oncogenic fusion protein has altered molecular activities relative to wild-type Pax3,10 which are believed to contribute to ARMS tumor phenotypes.11 Patients diagnosed with PAX3-FOXO1-positive ARMS have a 4-year survival rate of 8%12 which stems from the chemoresistance of metastatic tumors combined with a current lack of effective therapies specific for targeting ARMS. This information highlights the necessity of understanding the underlying biological and biochemical processes that contribute to the genesis of ARMS to develop much needed therapeutic alternatives.Rhabdomyosarcoma (RMS), one of the most common solid tumors in children,14 We published that wild-type Pax3 is phosphorylated at Ser201 and Ser205 by the kinases GSK3β and CK2, respectively.16 Upon the induction of differentiation, phosphorylation at Ser201 persists. However, phosphorylation at Ser205 is rapidly lost with a concomitant increase in phosphorylation on Ser209, again mediated by CK2.17 In contrast, we found that PAX3-FOXO1 is phosphorylated on Ser201 and Ser205 during proliferation; this status remains unaltered throughout myogenesis with no increase in phosphorylation at Ser209.16 Therefore, the aberrant phosphorylation of PAX3-FOXO1 may affect normal myogenesis to contribute the development of ARMS.Posttranslational modifications such as phosphorylation are common mechanisms for the regulation of transcription factors. As such, inhibition of these phosphorylation events provides an attractive target for drug development.in vitro cellular model,18 altered its transcriptional activity. Others demonstrated that small molecule inhibitors of GSK3β affected the viability and transformation capabilities of an ARMS tumor cell line.19 However, the first study utilized a general mutation approach that altered several serine residues within a region without specifically targeting the known sites, whereas the second study failed to demonstrate that the small molecule inhibitors directly altered phosphorylation of PAX3-FOXO1. Further, neither of these studies demonstrated a direct dependence of biological outcomes on alterations of the specific and identified PAX3-FOXO1 phosphorylation events. Finally, PAX3-FOXO1 phosphorylation has yet to be studied in human primary ARMS tumor samples. Therefore, we wished to determine how inhibiting specific sites of PAX3-FOXO1 phosphorylation affects known ARMS tumor phenotypes and how these biological effects correlate to primary tumor samples to identify potential ARMS-specific biological targets for future therapy development.Previous work demonstrated that inhibiting phosphorylation of PAX3-FOXO1 in T-antigen-transformed human embryonic kidney cells (293T cells), a non-physiologically relevant In this study, we utilize small molecule inhibitors of GSK3β or phospho-incompetent mutations that individually target the known sites of phosphorylation to determine how inhibiting these events on PAX3-FOXO1 affects ARMS tumor phenotypes. Our results demonstrate that inhibitors of GSK3β reduce phosphorylation of PAX3-FOXO1 at Ser201 and that inhibition of this event, either through small molecule inhibitors or mutational analysis reduces migration, invasion and proliferation in two independent ARMS tumor cell lines. Further, inhibition of phosphorylation at Ser205 by mutational analysis reduces ARMS tumor cell proliferation and anchorage-independent growth. Finally, we demonstrate that phosphorylation of PAX3-FOXO1 at Ser201 and Ser205 are present in human primary ARMS tumors and in cells that infiltrate the surrounding normal tissue. Taken together, our results support the idea that phosphorylation of PAX3-FOXO1 is an important contributor to ARMS tumor phenotypes and as such could be a key biological target for ARMS therapy development.19 We determined that GSK3β phosphorylates PAX3-FOXO1 at Ser201 in primary myoblasts and in ARMS cell lines.16 However, no studies correlated the inhibition of GSK3β with a decrease in phosphorylation at Ser201 or how these changes affect ARMS tumor phenotypes. Therefore, we incubated the ARMS tumor cell lines RH30 or RH4 with increasing concentrations of the commonly used GSK3β inhibitor lithium chloride 20 or the highly specific inhibitor AR-A014418 (IC50=10 μM).21 We utilized concentrations commonly used in published reports and that were demonstrated to be effective at inhibiting GSK3β in vivo.23 We determined the level of phosphorylation at Ser201 on endogenous PAX3-FOXO1 by western blot analysis using our anti-Pax3(pSer201) antibody.16 After normalization for total PAX3-FOXO1, we observed a titratable decrease in phosphorylation at Ser201 with increasing concentrations of both inhibitors in both cell lines  preventing use of these small molecule inhibitors in these studies. Therefore, to determine how specifically inhibiting PAX3-FOXO1 phosphorylation affects ARMS tumor phenotypes we utilized mutants in which each individual site was mutated to a phospho-incompetent alanine . The two ARMS cell lines were stably transduced, selected with puromycin, and selected cells were harvested from three independent transductions and pooled resulting in a single population for each individual mutant. By utilizing a population of transduced cells, we remove the potential for variability that may occur from clonal effects. An immunoprecipitation-western blot analysis demonstrated the ectopic expression of all mutants, with lower levels of expression being seen in the RH4 tumor cell line ; therefore, we were only able to utilize RH30 cells for these experiments. We observed a titratable decrease in invasive capacity with increasing concentrations of both LiCl and AR-A014418, resulting in a nearly 80% and 50% decrease, respectively  resulted in a level of cell death that precluded the use of these inhibitors in our proliferation assay (data not shown). Therefore, to determine the effect specifically inhibiting PAX3-FOXO1 phosphorylation has on proliferation, we used RH30 and RH4 cells stably transduced with PAX3-FOXO1 or the individual PAX3-FOXO1 phosphomutants. We saw no significant changes in the doubling time of RH30 cells stably transduced with wild-type PAX3-FOXO1 relative to the empty vector negative control. However, the stable expression of the phospho-incompetent S201A, S205A or S209A resulted in an increased doubling time from 35 h to nearly 50 h  chromosomal translocation (data not shown). Although the presence of ARMS in pulmonary tissue is rare,29 this sample provides the perfect internal negative control because the surrounding normal lung tissue does not express PAX3 or PAX3-FOXO1.To date, studies examining the direct role that phosphorylation of PAX3-FOXO1 plays in myogenesis and ARMS tumor development have been predominantly limited to 16 we found a majority of the tumor stained positive for phosphorylation at Ser201 and that this staining is enriched around the periphery of the tumor , penicillin G (200 U/ml) and streptomycin (200 μg/ml)) and grown in a humidified incubation chamber at 37 °C in 5% CO46 Phospho-specific point mutants in which the indicated serine was individually mutated to the phospho-incompetent alanine  were created by PCR amplification using previously created Pax3 point mutants as the template and primers containing a BamHI restriction sequence at the 3'-end. All constructs were confirmed by sequencing.PAX3-FOXO1 was cloned into the modified, low-copy number retroviral expression vector MSCV-IRES-Puro, which contains the backbone of the low copy pSMART GC LK vector . We introduced a single nucleotide silent mutation, thereby creating a novel BamHI site in PAX3-FOXO1 that facilitated the cloning of phospho-incompetent mutants. The presence of the IRES allows the dual expression of PAX3-FOXO1 and the puromycin resistance gene as a single transcript, both under control of the constitutively active Murine Stem Cell Virus promoter.17 Retroviral stocks were generated by the transient transfection of 293T cells with 3 μg each of retroviral packaging vectors pSRα-G and p-EQ-PAM-E and 3 μg of the above-described expression vectors using the TransIT-LT1 reagent . Culture supernatants containing virus were collected between 36 and 72 h after transfection, filtered (0.45 μm) and subsequently used for a single transduction, as previously described.17 Two to 3 days post transduction, transduced cells were selected by incubation in media supplemented with puromycin (2.5 ng/ml) until complete cell death was observed in the non-transduced control cells.ARMS tumor cells were stably transduced with the empty vector, wild-type PAX3-FOXO1 or PAX3-FOXO1 phosphomutants  as previously described.47 Western blot analysis of endogenous PAX3-FOXO1: non-transduced RH30 or RH4 cells were grown to approximately 80% confluency. Growth media was replaced with media containing increasing concentrations of the GSK3β inhibitors LiCl  or AR-A014418  and incubated for 24 h at 37 °C and 5% CO2. Alternatively, stably transduced cells were grown to 80% confluency in the absence of any inhibitors and harvested. Equal amounts of total cell lysates (25 μg) were separated by 8% SDS–PAGE and the analysis was performed as previously described.16Antibodies specific for PAX3-FOXO1 (anti-Pax3) or PAX3-FOXO1 phosphorylated at Ser201 (anti-Pax3[p201]) were produced as described previously.M, 20 mM or 40 mM) or AR-A014418  and incubated for 24 h at 37 °C and 5% CO2. Alternatively, stably transduced RH30 or RH4 cells were seeded onto a 24-well plate at 300 000 cells per well. A 100 μl gel-loading tip was used to generate a scratch in the monolayer, the cells were carefully washed twice with 1 × phosphate-buffered saline to remove floating cells, and subsequently incubated with fresh media containing the GSK3β inhibitors or non-supplemented media, respectively. Images were captured at exact spots every 15 min for 6 h using an Olympus time-lapse microscope  with subsequent analysis using SlideBook software. Sixty to 80 individual cells from four independent images were tracked and the velocity was determined for each cell using ImageJ software.Non-transduced RH30 or RH4 cells were seeded onto a 24-well plate at 200 000 cells per well. Growth media was replaced with media containing increasing concentrations of LiCl  at 25 ng/ml and the respective inhibitors in the lower plate. The invasive potential of the stably transduced RH30 cells was determined as just described, except all incubations were performed in the absence of inhibitors. All samples were determined in triplicate and P-values were computed using non-parametric two-way analysis of variance.Non-transduced RH30 cells were grown on 100mm dishes until approximately 70% confluent, at which time they were incubated in the presence of GSK3β inhibitors at the concentrations described above. After 24 h, the cells were harvested and the invasive potential was determined using the BD Biocoat Tumor Invasion System  as previously described°C in the presence of 5% CO2. Color formation was detected by measuring the absorbance at 450 nm using a microplate reader, plotted as a function of time, and doubling times calculated using GraphPad Prism 6 software. P-values were computed using non-parametric two-way analysis of variance.The proliferation rate of stably transduced RH30 and RH4 cells was assessed using a CCK-8 colorimetric assay kit . Cells were seeded at a density of 25 000 cells per well in a 24-well plate and allowed to grow for up to 7 days. The assay was conducted in triplicate, with an independent well being used for each replicate for each day of cellular growth. On each day of growth, 50 μl of CCK-8 solution was added to the individual well being tested and incubated for 2 h in a humidified chamber at 37 P-values were computed using non-parametric two-way analyss of variance.In separate experiments, once reaching 100% confluence, stably transduced RH4 cells were allowed to grow an additional 5 days to determine their foci forming capacity. Total numbers of foci formed were counted, all samples were determined in quadruplicate, and 2. Transformation and colony formation was determined using the CyQuant GR fluorescent dye according to the protocol provided by the manufacturer.Anchorage-independent growth assays were performed using a CytoSelect 96-well cell transformation assay kit . Stably transduced RH30 cells were plated in 1.2% agar in a 96-well plate at 5000 cells/well and cultured for 8 days at 37 °C and 5% CO2O. The sections were loaded onto the Ventana Ultra IHC stainer  and prepared according to the manufacturer's specifications. Briefly, slides (tissues) were treated with Cell Conditioner  at 95 °C for 92 min. After rinsing with reaction buffer, the slides were warmed to 36 °C. I-View Inhibitor was applied for 4 min; the slides were rinsed using reaction buffer. The sections were then incubated with antibodies specific for Pax3 or Pax3 phosphorylated at Ser201 or Ser205 for 2 h at 36 °C. After incubation, all slides were treated with Ventana's Blocker A-B solution for 8 min. The slides were rinsed with reaction buffer; I-View Biotin Ig was applied for 8 min. After rinsing with reaction buffer, the slides were treated with I-View SA-HRP for 8 min, rinsed with reaction buffer. I-View DAB and I-View H202 was applied to the slides for 8 min after which the slides were rinsed in reaction buffer. The slides were treated with I-View Copper for 4 min, rinsed with reaction buffer, stained for 4 min with hematoxylin and toned with Bluing reagent for 4 min. Once the reactions were completed, the sections were washed in soapy distilled H2O, dehydrated , cleared (xylene) and cover-slipped.Four micrometer-thick sections of human primary ARMS tumor were mounted on glass slides and incubated in an oven overnight at 65 °C to ensure tight adherence of tissue to the slides. Deparaffinization was carried out using xylene, graded alcohols and distilled H