Glucose oxidase in the presence of glucose generated H2O2, which acted as initiator of polymerization reaction. The influence of polymerization bulk composition and pH on the formation of PANI- and Ppy-based nanocomposites was investigated spectrophotometrically. The highest formation rate of PANI- and Ppy-based nanocomposites with embedded glucose oxidase and gold nanoparticles  was observed in the solution of sodium acetate buffer, pH 6.0. It was determined that the presence of AuNPs or AuCl4− ions facilitate enzymatic polymerization of aniline and pyrrole.Several types of polyaniline (PANI) and polypyrrole (Ppy) nanocomposites with embedded glucose oxidase (GOx) and gold nanoparticles (AuNPs) were formed by enzymatic polymerization of corresponding monomers (aniline and pyrrole) in the presence of 6 and 13 nm diameter colloidal gold nanoparticles (AuNPs Intensive studies and the application of conducting polymers (CPs), such as polyaniline (PANI) and polypyrrole (Ppy), have been developed extensively over the past few decades ,2,3,4,5.2O2, which is produced during glucose oxidase catalysed oxidation of glucose [Usually, CPs are synthesized by chemical ,22,23,24 glucose ,9,28,31. glucose ,20,30,31Hybrid materials based on CPs containing inorganic nanoparticles  have unique properties and, therefore, they are promising for the application in electronics, optoelectronics and in bio-devices ,35,36,37In this research enzymatic polymerization based formation of polyaniline and polypyrrole nanocomposites with embedded glucose oxidase and gold nanoparticles is described. Polyaniline and polypyrrole based composite formation rate in the presence of different AuNPs, at different pHs of solution was evaluated by UV/Vis spectroscopy. Aspergillus niger, 201 units mg−1 protein) and D-(+)-glucose were purchased from Fluka  and Carl Roth GmbH+Co.KG , respectively. Before the investigations glucose solution was allowed to stay overnight for the formation of equilibrium between α and β optical isomers. Tetrachloroauric acid (HAuCl4·3 H2O) was obtained from Alfa Aesar GmbH&Co KG , tannic acid – from Carl Roth GmbH + Co  and sodium citrate – from Penta . All solutions were prepared using deionized water purified with water purification system Millipore S.A. . The solution of sodium acetate (SA) buffer (0.05 mol L−1 CH3COONa·3H2O) with 0.1 mol L−1 KCl was prepared by mixing of sodium acetate trihydrate and potassium chloride, which were obtained from Reanal  and Lachema . Aniline and sodium hydroxide (NaOH) were purchased from Merck KGaA , pyrrole – from Acros Organics  and hydrochloric acid (HCl) – from Sigma-Aldrich . All chemicals used in experiments were either analytically pure or of highest quality. Polymers were filtered before each measurement through 5 cm column filled by Al2O3 powder to remove coloured components. All solutions were stored between measurements at +4 °C. The synthesis of AuNPs(6nm) and AuNPs(13nm) was performed according to methodology reported previously [Glucose oxidase  or AuNPs(13nm) or 0.6 mmol L−1 of HAuCl4. Formed nanocomposites were separated from the synthesis solution by a centrifugation , then they were 2 times washed with 0.05 mol L−1 sodium acetate, pH 6.0 and were collected by centrifugation. Separated and washed PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposites were re-suspended in SA buffer, pH 6.0 and used for further investigations, which were performed at room temperature. PANI/AuNPs-GOx and Ppy/AuNPs-GOx composites were formed at room temperature (+20 ± 2 °C) in darkness in solution containing 0.05 mol L4−) were evaluated after enzymatic formation of nanocomposites in 0.05 mol L−1 SA buffer, pH 6.0, 0.05 mol L−1 of glucose, 0.50 mol L−1 of aniline or pyrrole, 0.75 mg mL−1 of GOx and 26.0, 3.6 nmol L−1 of AuNPs(6nm) or AuNPs(13nm) or 0.6 mmol L−1 of HAuCl4.The presence of gold in the polymerization solution, the pH of medium and the time of enzymatic polymerization have an influence on the synthesis of polymer’s nanoparticles. The investigations of the influence of AuNPs or chloroaurate ions , AuNPs(13nm) or AuCl4− was studied at different pHs ranging from 1.0 to 12 after 2 days. 0.1, 0.01 or 0.001 mol L−1 of HCl, 0.05 mol L−1 of SA buffer, pH 6.0, and 1 × 10−6, 1 × 10−4 or 0.01 mol L−1 of NaOH solutions were used for the preparation polymerization bulk solutions in pH range between 1.0 and 12.0. The duration of polymerization reaction was from 5 hours until 12 days in 0.05 mol L–1 SA buffer, pH 6.0, 0.05 mol L−1 of glucose, 0.50 mol L−1 of aniline or pyrrole, 0.75 mg mL–1 of GOx and 26.0 nmol L–1 of AuNPs(6nm) or 0.6 mmol L−1 HAuCl4. The polymerization in bulk solution consisting 0.05 mol LThe polymerization course was evaluated by UV/Vis spectroscopy. Optical absorbance of PANI/AuNPs-GOx and Ppy/AuNPs-GOx solutions was monitored by UV/Vis spectrometer Lambda 25  at the wave length range of 233–1000 nm. The investigations were performed at room temperature in plastic disposable cuvettes of 1 cm optical path length by the registration of UV/Vis spectra. After 5–10 minutes after the engagement of polymerization reaction at +20 ± 2 °C optical absorbance of PANI/AuNPs-GOx and Ppy/AuNPs-GOx solutions was registered. SigmaPlot software 12.5 was used to evaluate the data of UV/Vis measurements. The main steps of PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposite formation during enzymatic polymerization are presented in CPs such as polyaniline and polypyrrole are often used in catalysis ,20 or in(6nm) and AuNPs(13nm) in SA, pH 6.0, any optical absorbance peaks were registered in vis-spectra. Although, in the presence of AuCl4− instead of AuNPs the solution of pyrrole almost instantly becomes intensively black, what is clear evidence of polymerization reaction. As it is seen from (6nm), AuNPs(13nm) or HAuCl4 become brown (in the presence of PANI) and black (in the presence of Ppy) sediments. Such colours are the most characteristic for the delocalised π-electron systems of CPs [After the mixing of all reagents used for PANI and Ppy formation in the presence of AuNPss of CPs . λ = 285 nm and λ = 570 nm if AuNPs(6nm) are added into polymerization bulk solution and only one wide peak at λ = 550 nm appears if AuNPs(13nm) are added into polymerization bulk solution. If polymerization is performed in the presence of AuCl4− the peak maximum appears at λ = 450 nm. This fact is an agreement with other researches [λ = 330 nm appears due to π-π* transition of the benzenoid ring and another peak at λ = 440 nm—due to polaron π-transition [λ = 330 nm is indication of 5,10- dihydrophenazin formation, which is cyclic aniline dimer [π-π* transition is associated with the delocalization of electrons from benzene rings on nitrogen atoms of aniline [λ = 436 nm. It is due to electron‘s excitation of benzenoid highest occupied molecular orbital to quinoid lowest unoccupied molecular orbital and indicates the formation of emeraldine forms of PANI [The intensity of colour of synthesized polymers also depends on the pH of initial polymerization bulk solution. The range of tested pHs was between 1.0 and 12. As it is seen from  if AuNPsnm are adsearches ,37. As i if AuNPsnm are adansition . Resultsne dimer . π-π* tr if AuNPsnm are ad aniline ,29. The  if AuNPsnm are ad if AuNPsnm are ad of PANI ,24,29. T of PANI .λ = 570 nm. By the increase of pH value of polymerization solution, the polaron-related peak at λ = 420 nm gradually disappears and a strong absorbance due to excision transition of the quinoid rings at λ = 560–600 nm is emerging. At the same time in strong alkaline polymerization solution optical absorbance at λ = 257 nm and λ = 320 nm, which is observed due to ð-ð* transitions of the benzenoid rings, has increased [λ = 442–570 nm and the peak at λ = 450 nm in the presence of AuNPs and AuCl4–, respectively, were evaluated to assess the formation of PANI/AuNPs-GOx nanocomposites during the polymerization. Synthesized PANI/AuNPs-GOx nanocomposites were formed in the E-PANI, which has been confirmed by the appearance of brown colour.As it is seen from spectra presented in ncreased . The abs+ ions are produced during polymerization reaction. As seen from the presented curves ((6nm) were present in the polymerization solution; and from 0.256 to 0.223 a.u. in the case where AuNPs(13nm) were present in the polymerization solution and from 0.813 to 0.283 a.u. in the case where AuCl4− was present in the polymerization solution. Synthesis of some branched polyaniline chains is expected due to polaron transitions, which are reflected in spectra [(6nm) or AuCl4−, respectively. When the pH of polymerization solution containing AuNPs(13nm) was equal to pH 10, then optical absorbance decreased until 0.217.The influence of pH value of polymerization solution on synthesized PANI/AuNPs-GOx nanocomposites is presented in d curves E, the in spectra . When th4− in pH range from 2.0 to 12. Meantime, the auto-polymerization of PANI in the presence of AuNPs(6nm) and AuNPs(13nm) was observed only in pH ranges from 1.0 until 3.0. The auto-polymerization of aniline in polymerization solution of pH 2.0 with AuNPs(6nm), AuNPs(13nm) or AuCl4− was 1.83, 1.79 and 2.78 times slower than that in polymerization solution with the same gold-based compounds and GOx. For enzymatic formation of PANI/AuNPs-GOx nanocomposites neutral polymerization solution is required due to best enzymatic activity and stability of GOx [In research performed by another authors it was predicated that aniline in polymerization bulk solution remain mobile and the synthesis of polymers could be proceeded without any enzyme . Moreove4− ions were intensively black coloured and solutions above a sediment consisting of aggregated Ppy/AuNPs-GOx become dark green. Dark colour of formed Ppy-based nanocomposites is characterized for advanced π-conjugated system [λ = 265 nm, λ = 312–361 nm, λ = 414 nm and λ = 560 nm, what is in an agreement with spectra of chemically synthesized polypyrrole [λ = 265 nm is associated with the π-conjugated structure of pyrrole rings, at λ = 312–361 nm and at λ = 414 nm—with π-π* transition of the polypyrrole‘s chain [λ = 560 nm is attributed to a transition from the valence bond to the antibonding polaron- or bipolaron-state and reveals that ultra-small clusters of AuNPs and Ppy are formed [λ = 265 nm and λ = 312–361 nm was changed just slightly but at λ = 414 nm and λ= 560 nm changes of optical absorbance were very significant. The absorbance at λ = 414 nm is characteristic for the early stage of pyrrole oxidation.The influence of pH value of polymerization solution on optical absorbance during the formation of Ppy/AuNPs-GOx nanocomposites was investigated in the next stage of our investigations. During 2-days lasting enzymatic formation of Ppy/AuNPs-GOx nanocomposites at pH values ranging from 1.0 until 12 in the presence of AuNPs or AuCld system . As it iypyrrole ,33,34. T‘s chain ,33,34. Te formed ,33,34. Bλ = 480 nm has been changed from 0.230 to 0.403 a.u. in the presence of AuNPs(6nm), from 1.33 × 10−3 to 0.141 a.u. – of AuNPs(13nm) and from 0.248 until 0.608 a.u.– of AuCl4−. In polymerization solution of pH 12, the optical absorbance at λ = 480 nm decreased down until 0.169, 3.04 × 10−3 and 0.144 a.u. in the case of AuNPs(6nm), AuNPs(13nm) and AuCl4−, respectively. Such decrease of optical absorbance by increasing pH until 12 is an agreement with investigations of Ppy chemical synthesis in polymerization bulk solution without any gold-based compounds [As it is seen from ompounds .4− into Au0 occurs very quickly and simultaneously darkening of the solution is observed [4− ions and in the absence of GOx was observed at pHs ranging from 2.0 to 3.0 during the formation of Ppy/AuNPs(6nm) and Ppy/AuNPs(13nm) nanocomposites and from 1.0 to 3.0 during the formation of Ppy/AuNPs(AuCl4−) nanocomposites. The auto-polymerization of pyrrole in the presence of AuNPs(6nm), AuNPs(13nm) and AuCl4− in polymerization bulk solution at pH 2.0 was 5.47, 1.31 and 2.89 times slower, if compared with the enzymatic formation of Ppy/AuNPs-GOx nanocomposites.When polymerization compounds are mixed, then the polymerization of pyrrole and the reduction of AuClobserved . Similarobserved . It was Significant differences of the optical absorbance were detected in the absence and in the presence of GOx, what revealed a high impact of the enzyme to the polymerization rate of monomers . Changes(6nm), AuNPs(13nm) or AuCl4− ions. AuNPs are forming seeds for polymerization and AuCl4− ions are acting as oxidators, which initiates the polymerization of CPs [0 forms AuNPs, which act as seeds for polymerization. In the presence of HAuCl4 the reduction of AuCl4− and the formation of oligoaniline is the dominant reaction in the polymerization bulk solution [4− ions and PANI is following by next steps. First of all, polyaniline coordinates Au(III) via an amine N, which is substituting Cl− in AuCl4−, because four Cl− anions are released into polymerization solution during PANI formation [2− ion is able to coordinate with an imine N of the quinoid group through electron donation from N to Au and formation of N–H----Cl− type hydrogen bonds, therefore AuCl4− ion is taking active and important role in the formation of PANI/AuNPs based nanocomposites [Enzymatic formation of PANI/AuNPs-GOx and Ppy/AuNPs-GOx depends on source of gold, which was AuNPsn of CPs  and formm), AuNPsnm or AuCm), AuNPsnm or AuCormation . Then, Amposites .−1 sodium acetate buffer, pH 6.0, which was chosen as the most optimal for mentioned gold/polymers enzymatic polymerization. Higher value of optical absorbance of polyaniline was characterized for polymerization solution with AuCl4− and achieved 0.674 a.u. ((6nm) and AuNPs(13nm) the polymerization rate of polyaniline formation was very similar and optical absorbance of 0.274 and 0.256 a.u was registered, respectively. In the presence of AuCl4− the formation of PANI/AuNPs(AuCl4−)-GOx nanocomposites was 2.46 and 2.63 times faster than that in polymerization bulk solution containing AuNPs(6nm) and AuNPs(13nm). The same influence of gold-based compounds was observed during the polymerization of pyrrole. As it is presented in (AuCl4−)-GOx nanocomposites (0.608 a.u.) was 1.51 and 4.31 times higher if compared with results obtained in polymerization bulk solution containing AuNPs(6nm) (0.403 a.u.) and AuNPs(13nm) (0.141 a.u.). It is seen that the formation rate of PANI/AuNPs(AuCl4−)-GOx and PANI/AuNPs(13nm)-GOx nanocomposites was 1.11 and 1.82 times higher if compared with of Ppy/AuNPs(AuCl4−)-GOx and Ppy/AuNPs(13nm)-GOx ((6nm)-GOx formed nanocomposites colloidal solution was 1.47 times lower than that of Ppy/AuNPs(6nm)-GOx nanocomposites colloidal solution. It could be explained by short duration of polymerization.The influence of gold compounds and polymerization rate of polyaniline and polypyrrole nanocomposites formation was investigated in 0.05 mol L674 a.u. . However3nm)-GOx . Meantim4(AuCl−)-GOx and Ppy/AuNPs4(AuCl−)-GOx nanocomposites was 2.14 and 3.52 times higher if compared with that registered for PANI/GOx (0.315 a.u) [(6nm)-GOx and PANI/GOx was very similar; for Ppy/AuNPs(6nm)-GOx was 2.33 times higher than that obtained for Ppy/GOx nanocomposites. The formation rate of PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposites in comparison to that of PANI/GOx and Ppy/GOx is significantly higher, because AuNPs speed-up the agglomeration of formed oligomers and the most probably facilitate polymerization reaction [(6nm) and AuCl4− were chosen, which were both considered to be the most suitable for the formation of these aniline- and pyrrole-based nanocomposites [During control experiments we tested: (i) the polymerization bulk solutions of PANI and Ppy, which did not contain AuNPs and (ii) the solution, which contained glucose, GOx, and AuNPs but did not contain any monomers (aniline or pyrrole), which are required for the formation of polymers. As it is presented in 315 a.u)  and Ppy/reaction ,37. As imposites .(6nm) or 0.6 mmol L−1 AuCl4−, correspondingly. Visible aggregation of polyaniline and polypyrrole in the presence of AuNPs and AuCl4− were observed after 2 and 4.5 days of enzymatic polymerization. One of the most important parameters of polymerization process is the duration of the polymerization. Thus, the variation of duration of enzymatic polymerization enabled to achieve the optimal polymerization performance. The spectrometric investigation of enzymatic polymerization duration on formation of PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposites was performed in SA buffer, pH 6.0, in the presence of aniline or pyrrole and of AuNPsλ = 450 nm was achieved after 2 and 1 days for PANI/AuNPs(6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx nanocomposites formation. This optical absorbance band appears due to polaron transition from benzenoid group at highest occupied molecular orbital to quinoid group at lowest unoccupied molecular orbital, which indicates presence of emeraldine form of PANI/AuNPs-GOx nanocomposites and is in agreement with results by another authors [As it is presented in  authors ,24,27,30th day of polymerization. It is indicated, that the highest value of optical absorbance for PANI/AuNPs(6nm)-GOx (1.05 a.u.) and PANI/AuNPs4(AuCl−)-GOx (1.24 a.u.) was achieved after 10 days lasting enzymatic polymerization. The value of optical absorbance was 11.1 and 3.17 times higher than that after 5 hours lasing polymerization, respectively for PANI/AuNPs(6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx. Moreover, long polymerization’s duration enables to synthesize larger PANI/AuNPs-GOx nanocomposites. Therefore, for the formation of smaller nanocomposites short duration of enzymatic polymerization is recommended. As it is seen from (6nm) and 0.6 mmol L−1 AuCl4− optical absorbance at λ = 450 nm increased from 0.095 to 0.517 a.u. and from 0.391 to 1.11 a.u, respectively. The increase of optical absorbance by 5.44 and 2.84 times after 5 days for PANI/AuNPs(6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx formed nanocomposite colloidal solutions was observed. The appearance of this peak in the presence of small nanocomposites in polymerization solution could be explained by fast movement of free electrons to the surface of AuNPs-based nanocomposites, during which electrons are scattered and quickly lose the coherence. In other words, the surface plasmon resonance bandwidth increases by the decrease of AuNPs size [As it is seen from NPs size .(6nm) or 0.6 mmol L−1 AuCl4− to the reactants UV/Vis spectra -GOx and Ppy/AuNPs4(AuCl−)-GOx nanocomposites were achieved at λ = 550 nm and λ = 590 nm, respectively. After one day of enzymatic polymerization, these waves almost disappeared and new bands of π-π* transition of Ppy/AuNPs chain and a polaron transition similar to that of an ordinary Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx at λ = 448 nm and λ = 460 nm were observed more clearly [(6nm) or 0.6 mmol L−1 AuCl4− in polymerization bulk solution, a dark pyrrole was relatively rapidly synthesized and optical absorbance of formed nanocomposite colloidal solution, which was measured at two polymerization periods (after 5 h and 10 days) increased from 0.137 to 0.781 a.u. and from 0.159 to 1.05 a.u., respectively. It is seen that optical absorbance, which is characteristic for Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl-)-GOx nanocomposites, after 10 days of polymerization was 5.70 and 6.60 times higher than that registered after 5 hours. This indicates the formation of polypyrrole oligomers [(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx nanocomposites has changed optical absorbance of polymerization bulk solution until 0.494 and 0.538 a.u, respectively. It was determined that after 4.5 days of enzymatic polymerization the value of optical absorbance for Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl-)-GOx nanocomposites increased by 3.61 and 3.38 times, respectively. The decrease of absorbance value after 12 days of formation of nanocomposites is explained by aggregation of nanocomposites due their limited solubility.Enzymatic polymerization of pyrrole started very fast and after 5 hours of initiation of polymerization by addition of AuNPs spectra D,E showe clearly ,28,33,34 clearly . It was ligomers . From 5 (6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx nanocomposites was 1.05 and 2.06 times higher compared with that of Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx.It was demonstrated that synthesized PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposites were similar to that obtained by chemical or electrochemical methods ,33,34 anV) was evaluated using formula V = (tgα), where tgα was calculated from (6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx; 1.73 and 2.23 a.u. for Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx) formed when 100% of the monomers present in the solution are converted into polymers. Initial polymerization duration in polymerization bulk solution containing aniline with AuNPs(6nm) or AuCl4− was determined as 0.0026 and 0.0022 mol L−1 day−1, respectively; for solution containing pyrrole with AuNPs(6nm) or AuCl4− – 0.0031 and 0.0029 mol L−1 day−1, respectively. Initial formation rate of PANI/AuNPs(6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx nanocomposites was 1.19 and 1.32 times slower than that of Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx, respectively. However, AuNPs do not form the uniform and well interconnected structure because nanocomposites are embedded between polymer backbone [The polymerization rate  and 0.6 mmol L−1 of AuCl4− are the most suitable for the enzymatic polymerization of PANI- and Ppy-based nanocomposites. The highest rate of PANI/AuNPs-GOx and Ppy/AuNPs-GOx formation was observed in the solution of SA buffer, pH 6.0. The most optimal duration of the enzymatic synthesis of nanocomposites was within 4.5 days. In present our research enzymatically formed nanocomposites are attractive, because any other chemical compounds except glucose, GOx, AuNPs or AuCl4− and corresponding monomer (aniline or pyrrole), which forms conducting polymer matrix, are used in the polymerization course.Enzymatic polymerization of aniline and pyrrole monomers in the presence of AuNPs and AuCl


In contrast to non-canonical ligands, canonical Wnts promote the stabilization of β-catenin, which is a prerequisite for formation of the TCF4/β-catenin transcriptional complex and activation of its target genes. This pathway is initiated by binding of Wnt ligands to the Frizzled/LRP5/6 receptor complex, and it increases the half-life of β-catenin by precluding the phosphorylation of β-catenin by GSK3 and its binding to the βTrCP1 ubiquitin ligase. Other intercellular signals are also activated by Wnt ligands that do not inhibit GSK3 and increase β-catenin protein but that either facilitate β-catenin transcriptional activity or stimulate other transcriptional factors that cooperate with it. In this review, we describe the layers of complexity of these signals and discuss their crosstalk with β-catenin in activation of transcriptional targets. The relevance of the Wnt/β-catenin pathway  has been well established in embryonic development and in diseases such as cancer . In a WnReceptor signaling is initiated by binding of the specific ligand and leads to the formation of protein aggregates known as signalosomes, which incorporate or release different factors along the progression of the pathway. Canonical Wnt signaling is initiated by the binding of factors such as Wnt3a or Wnt1 to a receptor complex comprising Fz and LRP5/6. As a first reaction, Wnt-induced assembly of the complex activates CK1ε by promoting the interaction of CK1ε, which is indirectly bound to LRP5/6 through E-cadherin/p120-catenin, with the Fz-associated PR61ε, a subunit of PP2A phosphatase that controls its activity a 7,8]. ,8. 7,8].2 ,1514,15]2 . PtdIns, GSK3 inhibition would be expected to modulate the half-life of many of its substrates, and not only β-catenin. Indeed, Wnt increases the stability of many proteins ,41. ThisEven though canonical and non-canonical Wnts have opposite effects on the level of β-catenin  , both caThe interaction between Fz2 and Fyn does not require Dvl2 and is incompatible with the association between Fz2 and Dvl2, further demonstrating that these two interactions (Fz2-Fyn and Fz2-Dvl2) define two distinct, mutually antagonistic arms of Wnt signaling  Figure . PreviouAlthough Fyn controls the transcription of many canonical Wnt target genes, full activation of the Wnt3a pathway depends not only on activation of Dvl2 but also of Src/Fyn/STAT3–dependent branch . This liSNAI1 . Ac. Ac74]. Matrilysin [The transcriptional factor kaiso inhibits the expression of many canonical Wnt target genes. This was initially attributed to its direct interaction with 5′-CTGCNA-3′ motifs present in Wnt target promoters, such as trilysin ,80,81. Htrilysin , and thetrilysin . Kaiso atrilysin . Finallytrilysin ,85,86. TCDKN2A , for the inactivation of negative cofactors (such as kaiso), and for the stimulation of additional transcriptional factors that cooperate with the β-catenin/TCF4 complex (such as STAT3). Moreover, Wnt signaling impacts the stability of other proteins, such as TAZ, indicating the pleomorphic effect of this pathway. Finally, these effects of canonical Wnt ligands are in some cases shared by other Wnts that stimulate the non-canonical pathway: for instance, both types of ligands stimulate Rac1 and STAT3. In any case, many different aspects of this transduction cascade remain to be clarified to better explain the crosstalk with other pathways and to fully characterize the molecular reactions underlying them.


Mutations at position K171 in the kinase activation loop of Inhibitor of κB kinase beta (IKKβ) occur in multiple myeloma, spleen marginal zone lymphoma and mantle cell lymphoma. Previously, we demonstrated that these result in constitutive kinase activation and stimulate Signal Transducer and Activator of Transcription 3 (STAT3). This work also identified K147 as a site of K63-linked regulatory ubiquitination required for activation of signaling pathways. We now present a more detailed analysis of ubiquitination sites together with a comprehensive examination of the signaling pathways activated by IKKβ K171E mutants. Downstream activation of STAT3 is dependent upon the activity of: UBE2N, the E2 ubiquitin ligase involved in K63-linked ubiquitination; TAK1 (MAP3K7), or TGFβ Activated Kinase, which forms a complex required for NFκB activation; JAK kinases, involved proximally in the phosphorylation of STAT transcription factors in response to inflammatory cytokines; and gp130, or IL-6 Receptor Subunit Beta which, upon binding IL-6 or other specific cytokines, undergoes homodimerization leading to activation of associated JAKs, resulting in STAT activation. We further demonstrate, using an IL-6-responsive cell line, that IKKβ K171E mutants stimulate the release of IL-6 activity into conditioned media. These results show that IKKβ K171E mutants trigger an autocrine loop in which IL-6 is secreted and binds to the IL-6 receptor complex gp130, resulting in JAK activation. Lastly, by examining the differential abundance of proteins associated with K63-only-ubiquitinated IKKβ K171E, proteomic analysis demonstrates the global activation of proliferative responses. As cancers harboring K171-mutated IKKβ are likely to also exhibit activated STAT3 and p44/42 MAPK (Erk1/2), this suggests the possibility of using MAPK (Erk1/2) and JAK inhibitors, or specific ubiquitination inhibitors. K63-linked ubiquitination occurs in other kinases at sites homologous to K147 in IKKβ, including K578 in BRAF V600E, which serves as an oncogenic driver in melanoma and other cancers. Many mutations in effectors and regulators of the nuclear factor kappaB (NFκB) signaling pathway have been identified in multiple myeloma, contributing to disease onset and viability . MutatioIKKβ is the master regulatory kinase that activates the NFκB inflammatory pathway via Ser/Thr phosphorylation of Inhibitor of κB (IκB) proteins, thus targeting IκB proteins for degradation leading to the release of NFκB for nuclear translocation. In response to inflammatory stimuli, Transforming Growth Factor-Beta-Activated Kinase 1 (TAK1) activates IKKβ by phosphorylating Ser177, which primes the autophosphorylation of Ser181 in IKKβ . IKKβ coIn this work, we present a more comprehensive examination of the signaling pathways activated by IKKβ K171E mutants, including a detailed analysis of ubiquitination sites. Downstream activation of STAT3 in response to IKKβ K171E mutants is dependent upon the activity of: 1) UBE2N, the E2 ubiquitin ligase involved in K63-linked ubiquitination; 2) TAK1 (MAP3K7), or TGFβ Activated Kinase, which forms a complex required for NFκB activation; 3) JAK kinases, involved proximally in the phosphorylation of STAT transcription factors in response to inflammatory cytokines; 4) gp130, or IL-6 Receptor Subunit Beta which, upon binding IL-6 or other specific cytokines, undergoes homodimerization leading to activation of associated JAKs, resulting in STAT activation. We further demonstrate, using an IL-6-responsive cell line, that IKKβ K171E mutants stimulate release into conditioned media of IL-6 activity. Lastly, by examining the differential abundance of proteins associated with K63-only-ubiquitinated IKKβ K171E, proteomic analysis demonstrates the global activation of proliferative responses.We previously utilized LC-MS/MS to show that IKKβ is ubiquitinated at K147, K301, K418, K555 and K703 and that among these, only K147 was required for the biological activity of the activated IKKβ . In the Mutations at K171 within the kinase activation domain of IKKβ have been identified in several hemotological malignancies –4, eitheConstructs 7, 8, 9). The mutation K147R significantly reduces the kinase activity.Lastly, these mutations were combined with a mutation at the previously identified major site of K63-linked ubiquitination in IKKβ , the mutThis collection of IKKβ constructs was examined for activation of IKKβ using an antiserum that detects phosphorylation of IKKβ within the activation loop . AlthougWe have previously utilized the phosphorylation of the signal transducing protein STAT3 to measure downstream signaling by activated IKKβ . As showWhen this panel of IKKβ mutants was examined for total ubiquitination, using coexpression with an HA-tagged ubiquitin to allow anti-HA immunoprecipitation, immunoblotting to detect ubiquitinated IKKβ  suggestsThe 500 Hats of Bartholomew Cubbins,” in which Bartholomew is repeatedly ordered to remove his hat only to have another one mysteriously appear, no further effort was made to remove additional sites of ubiquitination beyond the 9KR series of constructs in IKKβ.rd panel), the inhibition of K63-linked ubiquitination by NSC697923 showed no inhibitory effect. Thus, K63-linked ubiquitination is required for the downstream activation of STAT3 by IKKβ, but not for activation of IKKβ itself.The small-molecule inhibitor NSC697923 blocks the formation of K63-linked ubiquitin polymers that contribute to the activation of the IKK complex during inflammation-induced activation of NFκB –15. The th panel, Lane 4 vs. 9), whereas phosphorylation of IKKβ K171E was not . These results demonstrate a requirement for TAK1 in the downstream activation of STAT3 in response to activated IKKβ.IKKβ is typically activated by the upstream protein kinase TAK1 . (5Z)-7-st panel) shows the resulting inhibition of STAT3 phosphorylation in the presence of JAK Inhibitor 1 (Lanes 3–5 vs Lanes 9–11). In this experiment, we also showed that JAK Inhibitor 1 inhibited phosphorylation of STAT3 in response to exogenous IL-6 (Lane 13 vs 14).STAT3 is typically activated via IL-6-induced dimerization of the gp130 subunits of IL-6R, subsequently activating the JAKs to phosphorylate STAT3 . In ordest panel), conditioned media from cells expressing the K171E mutants–in the background of WT, 4KR, or 9KR–clearly stimulated phospho-STAT3, and this was completely blocked by the presence of SC144 (Lanes 3–5 vs Lanes 9–11). Again, as a control, SC144 completely inhibited the response to exogenously added IL-6 (Lane 13 vs 14).We also considered whether an autocrine signaling response to IL-6 was induced by IKKβ K171E mutants, a model which will be examined further below. The data presented in st and 2nd panels), in the absence or presence of NSC697923 to inhibit the E2 ubiquitin conjugating enzyme UBE2N (Lanes 3–5 vs Lanes 9–11). Although NSC697923 largely inhibits ubiquitination of IKKβ in this assay, it does not eliminate it completely. In the darker exposure, there is a very strong signal of polyubiquitinated IKKβ, indicated by the brackets, which is attenuated in the presence of the UBE2N inhibitor. These results demonstrate that the apparent ubiquitination of IKKβ K171E mutants occurs significantly by a K63-linked pathway, although not exclusively. Of note is the massive ubiquitination of the IKKβ K171E mutants in comparison with IKKβ WT, which is almost devoid of a ubiquitination signal except in the dark exposure .To further examine the ubiquitination of the IKKβ K171E mutants, we exploited an HA-tagged ubiquitin clone in which all Lys residues except for K63 were removed by mutagenesis. When coexpressed with IKKβ K171E proteins, this should result in a highly enriched signal for K63-linked ubiquitination. The results of this experiment are shown in rd panel), the inhibition of K63-linked ubiquitination by NSC697923 showed little inhibitory effect. Thus, in cells that are IKKβ-deficient, K63-linked ubiquitination is required for the downstream activation of STAT3 by IKKβ, but not for activation of IKKβ itself.To rule out the possibility that the presence of endogenous IKKβ might have a major effect in the preceding experiments, we also utilized IKKβ-deficient murine cells expressing IKKβ mutants. These were examined in the absence or presence of the small-molecule inhibitor NSC697923 to inhibit K63-linked ubiquitination. The effect of NSC697923 on activation of STAT3 is shown in The K171E mutation of IKKβ was initially identified in multiple myeloma  and spleExperiments with INA-6 cells, however, were much more revealing as presented in Overall, the data in In order to probe changes in the abundancy of total proteins and of ubiquitin-conjugated proteins as a consequence of activated IKKβ K171E mutants, we employed a proteomics approach. Large samples of 1.8x10e7 HEK293T cells were prepared expressing IKKβ WT or IKKβ K171E 9KR, in the presence of the HA-tagged K63-only-Ub. Four replicates of each sample were prepared. These lysates were immunoprecipitated with anti-HA mAB, subjected without further purification or enrichment by LC-MS/MS, and spectral data was then analyzed by MaxQuant label free quantitation (LFQ) . This apAs presented in We present experiments probing signaling by the oncogenic K171E mutation of IKKβ, originally identified in hematological malignancies –4. The KInitially, we had hoped we might create an IKKβ K171E mutant in which the only remaining site of ubiquitination would be K147. In this quest, we first eliminated K301, K418, K555, and K703, sites initially identified as ubiquitination sites, to create the 4KR mutants. Although this resulted in a slight relative increase in utilization of K147 for ubiquitination, many additional sites of ubiquitination were now observed. We then eliminated K310, K428, K509, K614, and K641 which, when combined with the 4KR mutants, created the 9KR mutants. Although K147 was now preferentially utilized, we still saw the appearance of many additional sites of ubiquitination. We found few changes in the ability of these different mutants to activate downstream pathways; as discussed below, IKKβ K171E, IKKβ K171E 4KR, and IKKβ K171E 9KR all exhibited relatively similar activation of downstream pathways.K63-linked ubiquitination stabilizes signaling functions of proteins and is an indispensable modification for the activation of innate and acquired immune responses, including DNA damage repair pathways . ActivatA model integrating our results in presented in We also employed a proteomics approach to compare cells expressing IKKβ WT in comparison with IKKβ K171E 9KR, examining all proteins recovered that were labeled with a coexpressed HA-tagged K63-only-Ub. These proteins were analyzed without further purification or enrichment by LC-MS/MS and MaxQuant label free quantitation . We founThe identification of IKKβ mutations at position 171 in hematological malignancies –4 suggesStarting with expression plasmids for IKKβ WT and IKKβ K171E , the derThe HA-Ub3 and HA-Nemo plasmids were described previously . The HA-2 at 37°C. Cells were transfected with plasmid DNA using calcium phosphate precipitation at 3% CO2. Approximately 18 h after transfection, cells were starved overnight before collecting in RIPA Lysis Buffer, as previously described [2 at 37°C. IKKβ knockout murine 3T3 cells [2.HEK293T were grown in DMEM with 10% FBS and maintained in 10% COT3 cells  were kin5 cells/ml in media lacking Marrow Max Bone Medium, containing the indicated concentrations of hIL-6. Samples of triplicate cultures were removed and examined by MTT metabolic assay at each time point.To determine the IL-6 concentration dependence, INA-6 cells were seeded at an initial density of 2.5 x 102 for 4 h, after which 500 μl of 0.04 M HCl in isopropanol was added and incubated again for at least 30 min. Absorbance was measured at 570 nm.For MTT metabolic assays, 500 μl of cultures were transferred in duplicate to a 24-well TC plate and incubated with 50 μl of 5 mg/mL of thiazolyl blue tetrazolium bromide (MTT) (Sigma) at 37°C in 5% CO4 cells/ml. FBS was added to sterile filtered starvation media prior to treatment at a 10% final concentration. Samples of triplicate cultures were removed and examined by MTT metabolic assay at each time point.IKKβ induced proliferation of INA-6 cells was determined by treating the cells with starvation media (RPMI1640) from HEK293T cells expressing IKKβ mutants. INA-6 cells were seeded at 3 x 105 cells/ml were lysed in RIPA after 48 h incubation with media from HEK293T cells expressing IKKβ plasmids, prepared as described above. Lysates were immunoblotted as described below. Triplicate immunoblots were quantitated.To determine STAT3 activation, INA-6 cells seeded at 1.4 x 10Lysates were collected in RIPA lysis buffer containing inhibitors 10 ng/ml Aprotinin, 1 mM PMSF, 1 mM Na3VO4, 20 mM β-glycerol-phosphate, and 5 mM N-ethyl-maleimide, and proteins were separated by 10% or 12.5% SDS-PAGE followed by transfer to Immobilon-P membrane. For immunoprecipitations, 400 μg of total protein was incubated with αHA (F7) overnight and collected with Protein A Sepharose (Sigma) or Pierce Protein A/B Magnetic Beads (Thermo Fisher Scientific). Immunoblotting reagents were from the following sources: antibodies against IKKβ (H-4), IKKβ (G-8), HA-probe (F-7), STAT3 (C-20), Myc (9E10), and TAK1 (M-579) from Santa Cruz Biotechnology; Phospho-IKKα/β (Ser176/180) (16A6), Phospho-STAT3 (Tyr705) (D3A7), and Phospho-TAK1 (T184/187) (4531S) from Cell Signaling Technology; HRP anti-mouse, HRP anti-rabbit, and Enhanced Chemiluminescence (ECL) reagents from GE Healthcare. Other reagents included: NSC697923 from Santa Cruz Biotechnology; JAK Inhibitor 1 from Calbiochem; MG132 and (5Z)-7-Oxozeaenol from Tocris Bioscience; SC144 from Sigma-Aldrich; recombinant human IL-6 from R&D Systems. IL-6 concentrations in conditioned media were determined by ELISA, using Human IL-6 Quantikine ELISA D6050 from R&D Systems according to the manufacturer’s protocol.3 and HA-NEMO plasmids. Cells were starved overnight and treated with 10 μM MG132 for at least 48 h before collection. Lysates were collected in RIPA and analyzed as previously described [For identification of ubiquitination sites, HEK293T cells were transfected with the indicated IKKβ plasmids together with HA-Ubescribed . For IKKescribed . For IKKescribed . Lysates18 Acclaim PepMap column 0.075 x 500 mm, 2 μm particles (Thermo Fisher Scientific) in a 94-min linear gradient of 2–28% solvent B at a flow rate of 300 nL/min. The mass spectrometer was operated in positive data-dependent acquisition mode. MS1 spectra were measured with a resolution of 70,000, an AGC target of 1e6 and a mass range from 350 to 1700 m/z. Up to 12 MS2 spectra per duty cycle were triggered, fragmented by HCD, and acquired with a resolution of 17,500 and an AGC target of 5e4, an isolation window of 1.6 m/z and a normalized collision energy of 25. Dynamic exclusion was enabled with duration of 25 sec.For the comparative analysis of IKKβ WT and IKKβ K171E 9KR, quadruplicate sets of 1.8x107 HEK293T cells were transfected, together with HA-K63-only-Ub. Lysates were sonicated, immunoprecipitated with anti-HA (F-7), and collected using Pierce Protein A/G Magnetic Beads. Dried samples were reconstituted with 2% acetonitrile, 0.1% formic acid and analyzed by LC-MS/MS using a Proxeon EASY nanoLC system (Thermo Fisher Scientific) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an analytical CLC-MS/MS spectra were analyzed using the quantitative proteomics software package MaxQuant which was used for peak detection, scoring of peptides, protein identification and label free quantification , 20. TheTo identify proteins with significantly different abundance between the two sample groups, a Student’s T-test, within the Perseus software, was performed on all proteins. An adjustment of the p-values for multiple testing was made using the Benjamini-Hochberg method. To identify significantly enriched pathways and functions (FDR < 0.05), the list of differentially abundant proteins was used as input into the WebGestalt package within the R statistical computing environment , 45. GenS1 TableList of differentially abundant proteins and Label Free Quantification (LFQ) values, raw and processed, for all proteins identified in mass spectrometry experiments. Differentially abundant protein list contains the results of statistical tests (with multiple testing correction) comparing abundance between WT and K171E 9KR IKKβ sample groups. Processing of LFQ data includes data imputation, width adjustment normalization, and log2 transformation.(XLSX)Click here for additional data file.S2 TableResults of functional and pathway enrichment analysis  performed on significantly differentially abundant proteins between WT and K171E 9KR IKKβ sample groups.(XLSX)Click here for additional data file.S3 TableList of proteins and quantified totals of ubiquitinated peptides in WT and K171E 9KR IKKβ sample groups sample identified in mass spectrometry experiments.(PPTX)Click here for additional data file.S4 TableRaw data for all identified proteins in all WT and K171E 9KR IKKβ samples including annotation, identified peptide totals, and label free quantification values.(XLSX)Click here for additional data file.


ERPs) were recorded in five sessions during somatosensory Go/No‐go paradigms  in an environmental chamber with temperature controlled at 20°C (Temperate) and 35°C (Hot). Increases in external canal temperature and heart rate were greater under the 35°C condition than under the 20°C condition. Regardless of thermal conditions, reaction times (RT) and error rates were not affected by the repetition of moderate exercise, whereas the peak amplitude of the N140 component, which is mainly related to somatosensory processing, was significantly reduced with the repetition of the exercise. However the peak amplitude of P300, which is linked to cognitive processes of context updating, context closure, and event‐categorization, was significantly smaller in post‐third and post‐fourth exercise bouts under the 35°C condition than under the 20°C condition, and this decrease was more prominent in No‐go trials under the 35°C condition. These results suggest that executive function, which is based on RTs and error rates, is not affected by prolonged exercise and different thermal conditions, whereas the exercise in a hot environment impairs human cognitive processing, particularly response inhibition.Cognitive performances may improve after acute moderate exercise, but not after prolonged and/or heavy exercise. The present study aimed to investigate the effects of environmental temperature during exercise on human cognitive processing. Fifteen healthy males performed four bouts of a 15‐min cycling exercise with a 10‐min rest between each bout, and event‐related potentials ( Exercise training or habitual physical activities elicit favorable changes in structural and functional brain networks , which was set to two different conditions . They performed the same absolute workload under both conditions. There was at least a 3‐day interval between the 20°C and 35°C conditions, and experiments were performed at the same time on a different day in a random order. On the first visit, exercise intensity was assessed in the anteroom under 20°C . Subjects wearing an electrocardiogram (ECG) monitoring device  sat on a cycle ergometer  in order to evaluate their workload. Exercise intensity was set to maintain a heart rate (HR) of approximately 130 bpm (~67% HR max) for 15 min of cycling in a temperate environment. The time interval between each bout of exercise was set at 10 min. A metronome sound was presented  to maintain smooth constant pedaling at 60 rpm. Exercise intensity was kept constant throughout the experiments under both conditions (132 ± 27 W). Subjects did not drink water throughout the experiment.On their arrival at the laboratory, subjects weighed themselves nude on a scale  with an empty bladder, and then wore only underwear and shorts. Subjects walked into the environmental chamber and rested quietly on a comfortable armchair for ~30 min. During the resting period, the following measurement equipment was attached. External canal temperature (Tear) was continuously measured using an infrared sensor  in the left ear canal, and sampled at 20 Hz via a data acquisition system . We previously confirmed that temperature measured using this device was reliable as an index of internal temperature  as quickly as possible only after the presentation of a Go stimulus. During recordings, subjects were instructed to keep their eyes open and look at a small fixation point positioned in front of them at a distance of approximately 1 m. One run comprised 80 epochs of a stimulation, which included 40 epochs for Go stimuli and 40 for No‐go stimuli. ERPs were recorded in five sessions during the somatosensory Go/No‐go paradigms  under 20°C (temperate) and 35°C (hot) conditions. EEG data were monitored but analyzed after the end of experiment. The practice session consisted of 10 stimuli before the recordings of the Pre session in each visit.μV. The analysis epoch for ERPs was 600 msec, including a prestimulus baseline period of 60 msec. Data were collected on a computer with a sampling rate of 1000 Hz. The peak amplitudes and latencies of N140 and P300 were measured at 110–210 and 240–500 msec, respectively. Amplitudes were measured baseline‐to‐peak. Reaction times (RT) were recorded on a computer, and slow responses exceeding 700 msec and incorrect responses were eliminated from averaging. N140 and P300 EEG values were obtained from the averaged responses of the Fz, Cz, Pz, C3, and C4 locations. The mean peak amplitude and latency for these variables, averaged across the five electrodes, were statistically evaluated via a three‐way ANOVA with repeated measures using within‐subjects factors of Condition (20°C vs. 35°C), Session , and Stimulus (Go vs. No‐go). Behavioral data on the mean RT, standard deviation (SD) of RT, and error rate including commission  and omission  errors were subjected to repeated measures ANOVAs with Condition and Session as factors. When a significant effect of Condition was observed, the post‐hoc paired t‐test was adjusted to identify differences between the 20°C and 35°C conditions. When a significant effect of Session was observed, the post‐hoc paired t‐test was adjusted to identify specific differences between the pre and other sessions.Each scalp electrode for EEG was referenced to linked earlobes. The ground electrode was placed at Fpz. Impedance was maintained at less than 5 kohm. The bandpass filter was 0.1–50 Hz. An electrooculogram (EOG) was also recorded to eliminate eye movements or blinks exceeding 100 A 60‐sec average was calculated for temperature variables  and HR before  and at the end of each exercise . Unfortunately, the Tear data logger did not record in three of fifteen subjects under either condition. Furthermore, the Tsk data logger did not record in two subjects. Since blood pressure was not measured at the end of exercise in some subjects, data obtained at rest and before each exercise were analyzed. These variables and mean blood pressure obtained before and at the end of each exercise were analyzed by a two‐way ANOVA with repeated measures using the within‐subject factors of Condition and Session. When the significant effects of Condition‐Session were identified, the post‐hoc Student–Newman–Keuls test was adjusted to identify specific differences . Significance was set at P < 0.001). ANOVAs revealed that Tsk was also significantly higher in the 35°C condition . ANOVAs revealed that HR was significantly higher in the 35°C condition . The results of the post‐hoc test are shown in Figure P < 0.01).Figure P < 0.05). This interaction revealed a difference in the mean RT between the 20°C and 35°C conditions with repeated sessions. Further analyses showed no significant main effects under the 20°C and 35°C conditions. Figure Figure n = 15). Black lines indicate waveforms under the 20°C condition, and gray lines show those under the 35°C condition. Figure P < 0.001). Post‐hoc testing for Session showed that the amplitude of Go‐N140 was significantly larger in pre than in the post‐ex4 session (P < 0.01) under the 20°C condition. The amplitude of No‐go‐N140 was significantly larger in the first than in the post‐ex3 (P < 0.05) and post‐ex4 sessions (P < 0.01) under the 20°C condition, and in the pre than in the post‐ex2 (P < 0.01), post‐ex3 (P < 0.05), and post‐ex4 sessions (P < 0.001) under the 35°C condition. These results suggested that the amplitude of N140 decreased with exercise repetition.Figure P < 0.05). The post‐hoc test for Condition in each Session showed that the peak amplitude of No‐go‐P300 was significantly smaller under the 35°C condition than under the 20°C condition in the fourth session (P < 0.05). ANOVAs for the peak latency of P300 revealed the significant main effects of Condition and Session . The post‐hoc test for Condition in each Session showed that the peak latency of Go‐P300 was significantly earlier under the 35°C condition than under the 20°C condition in the post‐ex3 session (P < 0.05). The post‐hoc test for Condition on each Session showed that the peak latency of No‐go‐P300 was significantly earlier under the 35°C condition than under the 20°C condition in the pre (P < 0.05), post‐ex1 (P < 0.01), post‐ex3 (P < 0.05), and post‐ex4 sessions (P < 0.05) . The latency of No‐go‐P300 was significantly later in the pre than in the post‐ex1 (P < 0.05) and post‐ex4 sessions (P < 0.05) during the 35°C condition  as well as RTs did not change in any sessions after each moderate exercise trial under the 20°C (temperate) condition. However, the neural activity of response inhibition  was gradually decreased under the 35°C (hot) condition, and was lower in the hot condition than in the temperate condition. Although the positive effects of acute exercise on cognitive processing were not observed in the temperate condition, the effects of repetitive exercises on cognitive function were observed when internal temperature markedly increased, particularly with the No‐go stimulus in the hot condition.Exercise increases circulating metabolites and hormones, which may enhance not only physiological function, but also psychological function. For example, catecholamines and brain‐derived neurotrophic factor (BDNF) have been implicated in cognitive performance  with the pressing of a button or counting events induced larger amplitudes at the parietal midline (Pz), but none or small amplitudes in standard stimuli. We previously reported greater reductions in the amplitudes of both P300 components under hyperthermic conditions and this impairment lasted longer in the No‐go‐P300 component than in the Go P300 component  and ventrolateral prefrontal cortices (VLPFC), supplementary motor area (SMA), primary sensorimotor area (SMI), anterior cingulate cortex (ACC), temporoparietal junction, temporal and parietal lobes, and thalamus  was used to measure ear canal temperature as an index of internal temperature. We recognized that this temperature has limitations as an index of internal temperature, but preferred to minimize any mental stresses associated with measurements. In our previous studies (Ogoh et al. The brain has the capacity for morphological and functional changes, and exercise training may elicit favorable changes in the structural and functional brain networks (Gomez‐Pinilla and Hillman The authors declare no conflicts of interest. The results of the present study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. The results of this study do not constitute endorsement by the American College of Sports Medicine.


KRAS mutant colon cancer but are met with significant resistance. Clinical investigations have demonstrated that these strategies are not well tolerated by patients.Mitogen-activated protein kinases (MEK 1/2) are central components of the RAS signalling pathway and are attractive targets for cancer therapy. These agents continue to be investigated in KRAS mutant colon cancers by LC-MS/MS analysis. We tested the MEK inhibitor in PIK3CA wild(wt) and mutant(mt) colon cancer cells. In addition, we tested the combinational effects of MEK and TNKS inhibitor in vitro and in vivo.We investigated a biomarker of response for MEK inhibition in PIK3CA wt colon cancer cells but not in mt. Tumour regression was promoted by combination of MEK inhibition and NVP-TNS656, which targets the WNT pathway. Furthermore, inhibition of MEK promoted tumour regression in colon cancer patient-derived xenograft models expressing PIK3CA wt.We identified β-catenin, a key mediator of the WNT pathway, in response to MEK inhibitor. MEK inhibition led to a decrease in β-catenin in PIK3CA mt colon cancer. Therefore, we suggest that β-catenin is a potential predictive marker of MEK inhibitor resistance.We propose that inhibition of the WNT pathway, particularly β-catenin, may bypass resistance to MEK inhibition in human  Colon cancer patients with KRAS mutations do not respond to cetuximab or panitumumab, which are antibodies that target epidermal growth factor receptor (EGFR).5 Because these mutations are found in 40% of colon cancers,6 additional treatment options and biomarkers of response are urgently needed for KRAS mutant cancers.Colon cancer has a high incidence, comprising ~13% of all cancers worldwide. Standard therapeutic approaches for treating colon cancer patients include combination regimens such as irinotecanKRAS. MEK inhibition has the potential to block inappropriate signal transduction regardless of the upstream position of the oncogenic aberration;7 in addition, MEK inhibition suppresses cell growth and induced apoptosis. In KRAS mutant cancers, the phosphatidylinositol 3-kinase (PI3K) genotype influences the patient’s sensitivity to MEK inhibitors.8PIK3CA mutations in various cancer cells correlate with resistance to MEK inhibitors, and cells transduced with PI3K mutant are resistant to MEK inhibition. KRAS stimulates multiple signalling effectors, including the PI3K pathway. Extensive crosstalk has been observed between the PI3K and RAS/RAF/MEK/ERK signalling pathways. Several studies have shown that the majority of MEK inhibitor-insensitive colon cancer cell lines harbour activating mutations in the PI3K pathway, whereas KRAS mutant cancer cells with an intact wild-type PI3K pathway are sensitive to MEK inhibitors.9Mitogen-activated protein kinase (MEK) is an essential component within the RAF/MEK/ERK pathway downstream of 10 Most phase I clinical trials exploring these combinations have been unable to increase the doses of either agent to the respective individual maximal tolerated dose.Recently, phase I clinical trials examined therapeutic approaches for the treatment of metastatic solid tumours using a combination of MEK inhibitors with PI3K/mTOR inhibitors.3 A key feature of the Wingless-INT (WNT) pathway is the regulated proteolysis of the downstream effector β-catenin by the β-catenin destruction complex. Constitutive β-catenin signalling due to either inactivating mutations in APC or activating mutations within β-catenin itself also plays a critical role in the development of colon cancer; nearly 90% of all colon cancers harbour mutations that drive β-catenin signalling.11 Several small molecules that target the WNT pathway have been developed, and their inhibitory effects on tumour growth have been reported.12 Tankyrase inhibitors (TNKSi) induce stabilisation of AXIN, which abrogates WNT/β-catenin signalling and induces apoptosis. Although many groups have studied WNT/β-catenin-targeted therapies, many important problems remain unsolved regarding inhibition of this pathway.The WNT/β-catenin pathway is associated with embryonic development and cancer progression, and its activation is highly prevalent in colon cancer.In our study, we attempted to identify a biomarker of MEK inhibition in colon cancer cells. First, we confirmed that the PI3K genotype is a key factor in determining sensitivity to MEK inhibitors. Second, we identified and evaluated β-catenin as a biomarker. We demonstrated that β-catenin plays a major role in the cell response to MEK inhibition. Moreover, combinational treatment with TNKSi and MEK inhibitors led to apoptosis in MEK inhibitor-resistant cells. Taken together, our results suggest that β-catenin is a novel predictive pharmacodynamic (PD) biomarker of MEK inhibitor resistance and a potential target for combinatorial treatment regimens.2. The DLD-1 isogenic cell lines PIK3CA wild-type (351) and PIK3CA mutant (353) were provided by Dr. Vogelstein, cultured in McCoy’s medium  supplemented with 10% FBS and penicillin/streptomycin (100 μg/ml), and maintained at 37 °C in an atmosphere containing 5% CO2. Additionally, the HCT116 isogenic cell lines β-catenin parent , β-catenin mt , and β-catenin wt  were provided by Dr. Vogelstein and cultured in McCoy’s medium supplemented with 10% FBS and penicillin/streptomycin.Human colon cancer cells were purchased from ATCC  or the Korea Cell Bank . The cells were cultured in RPMI medium or DMEM  supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 μg/ml)  and maintained at 37 °C in an atmosphere containing 5% COPI3K mutant: 353), and HCT116 isogenic cells  were plated and treated with 5 μM NVP-TNKS656 (tankyrase inhibitor) and 1 µM GSK112012 (MEK inhibitor). After 48 h, the cell death rate was determined with the Trypan blue exclusion assay.Cells were seeded and treated with the indicated dose of MEK inhibitor  . After 24 h, the cells were harvested and evaluated with the Trypan blue exclusion assay. Additionally, DLD-1, HCT-8, DLD-1 isogenic cells  or siRNA/shRNA were transfected with Lipofectamine 2000 or Lipofectamine RNAiMAX , as appropriate according to the manufacturer’s instructions. The sequence used in the siRNA/shRNA transfection studies is AACTATCAAGATGATGCAGAACTTGCC. shRNAs against β-catenin were acquired from Genolution .Clonogenic capacity was evaluated by plating vehicle- or MEK inhibitor-treated cells at a density of 100 cells per six-well plate. At 10–14 days after treatment, the cells were fixed and stained with 0.01% crystal violet solution.Cells were harvested and lysed in RIPA buffer , and the resultant proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PolyScreen membranes . The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and probed with antibodies targeting the following proteins: β-catenin , cyclin D1 , AXIN2, c-Myc, STAT3, cyclin B1, Gab1, c-caspase-3, p-ERK , HA, β-actin, and γ-tubulin (Santa Cruz Biotechnology). Primary antibodies were detected with HRP-conjugated anti-mouse or anti-rabbit antibodies, as appropriate, that were subjected to enhanced chemiluminescence .Cells were harvested, washed twice in PBS, and resuspended in 100 µl of annexin binding buffer (BD Pharmingen). Then, these cells were stained with 5 µl of annexin V and 1 µg/ml PI. After staining, 300 µl of annexin-binding buffer was added, and the stained cells were analysed using a flow cytometer (BD Bioscience).SW620 and DLD-1 cells cultured in the presence or absence of MEK inhibitor were harvested and lysed. After the protein concentration was detected with the BCA assay, SDS-PAGE, in-gel digestion, acidic/basic extraction, and DK-tip desalting for drug-treated cells were performed. Finally, the proteins were analysed with Triple TOP DDA or SWATH.3, the mice were administered the indicated doses of MEK inhibitor  for 28 days, and then the tumour volumes were monitored for 4 weeks (measured every 3 days). The tumour size was calculated as length × width2 × 0.5. After 28 days, the tumours were harvested, visualised, and measured. In addition, the tumours were analysed by western blot and immunohistochemistry, as described previously.13 In the patient-derived xenograft (PDX) model, tumour tissues were subcutaneously engrafted on the flank of 5-week-old female BALB/c nude mice. When tumour sizes reached 100 mm3, the mice were administered the indicated doses of MEK inhibitor  for 28 days. The tumour size and body weights were measured every 3 days for 4 weeks.Xenograft tumour growth in BALB/c nude mice was used as a model for in vivo experiments. All animal studies were conducted in compliance with animal protocols approved by the Asan Medical Centre Institutional Animal Care and Use Committee. SW620, DLD-1, and DLD-1 isogenic cells (351 and 353) were subcutaneously implanted. When tumour sizes reached 100 mmt-test, and the level of significance stated in the text was based on P values; a P value lower than 0.05 was considered statistically significant.All resulting data were statistically analysed using a two-tailed Student’s KRAS mutations, i.e., three PIK3CA wild-type cell lines and five mutant cell lines, to a variety of selective MEK inhibitors, i.e., AZD6244, AS703026, BAY 86–9766, and GSK112012.14 These cells were treated with these four MEK inhibitors at the indicated concentrations. Wild-type PIK3CA colon cancer cells displayed sensitivity to each of the MEK inhibitors; however, the mutant PIK3CA colon cancer cells did not respond to the MEK inhibitors  on MEK inhibitor-treated colon cancer cells that express mutant tor Fig. . Additiones Fig. . In addi2 Therefore, we investigated the combinatorial effect of a TNKS inhibitor NVP-TNKS656 with MEK inhibition in KRAS and PIK3CA mutant colon cancer cells. DLD-1 and HCT-8 cells, two colon cancer cell lines with mutant PIK3CA expression, were selected. Treatment with NVP-TNKS656 and the MEK inhibitors AZD6244 or GSK112012 significantly induced increases in cell death in both cell lines , which has the mutant allele knocked out, and DLD-1–PIK3CA mt (cell line 353), which has the wt allele knocked out. MEK inhibitor treatment of cell line 353 after transfection with scrambled shRNA did not induce cell death. In contrast, cell line 353 with β-catenin knockdown displayed increased cell death in response to MEK inhibitor  was orally administered to mice carrying these PDXs. Tumour growth and tumour weight decreased in the 52 PDX model following GSK112012 treatment  and NVP-TNKS656 (50 mg/kg), the latter of which targets the WNT pathway  cells. In addition, we demonstrated that β-catenin overexpression decreased cell death in MEK inhibitor-treated cells. Moreover, β-catenin expression was significantly decreased in the PIK3CA wild-type PDX model (#52) upon MEK inhibitor administration. In contrast, β-catenin expression did not change in the PIK3CA mutant PDX model upon MEK inhibitor administration. Thus, our results suggest that β-catenin is a predictive biomarker of MEK inhibitor sensitivity in colon cancer.Constitutive β-catenin signalling due to inactivating mutations in 20 In our study, we investigated possible approaches to overcoming MEK inhibitor resistance in colon cancer cells. We demonstrated β-catenin to be a key factor in the resistance of colon cancer cells to MEK inhibitors. β-Catenin was inhibited by the tankyrase inhibitor NVP-TNKS656, and combined treatment of NVP-TNKS656 and MEK inhibitor induced cell death in DLD-1 and HCT-8 (PIK3CA mt) cells. However, β-catenin mutant cells did not respond to the cotreatment of NVP-TNKS656 and MEK inhibitor. The combinatorial treatment also induced cell death in the DLD-1 isogenic PIK3CA mt cell line 353. In addition, we tested the combinatorial effects of GSK112012 and NVP-TNKS656 on HCT116-β-catenin isogenic cells, including 54 (parent as a control), 240 (β-catenin mt/mt) and 241 (β-catenin wt/-). NVP-TNKS656 alone and NVP-TNKS656/MEK inhibitor combination treatments induced cell death in 241 cells. However, neither 54 nor 240 responded to the same treatments (Supplementary Figure KRAS mutant and PIK3CA mt/β-catenin wt colon cancer cells.β-Catenin plays an important role in the WNT/β-catenin signalling pathway and is involved in both intracellular signalling and cell adhesion. Mutations of the β-catenin gene presumably disrupt these functions, leading to cell proliferation.KRAS mutant colon cancers. More importantly, combinatorial treatments such as NVP-TNKS656 and MEK inhibitor greatly induce cell death in a subset of KRAS mt/PIK3CA mt/β-catenin wt cells. Figure In conclusion, our study identified β-catenin signalling as an important factor in MEK inhibitor resistance in Supplementary Figure and Table legendSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6Supplementary Figure S7Supplementary Figure S8Supplementary Table S1Supplementary Table S2


Burkholderia mallei is the leading cause of glanders, a highly transmittable and an OIE-notifiable disease of equidae. Despite the importance of B. mallei, little is known about serodiagnosis of glanders. The present study aimed to develop an immunoblotting assay based on whole-cell proteome of B. mallei to enable accurate serodiagnosis of glanders.6 cfu/ml) of heat-inactivated B. mallei formulated with incomplete Freund’s adjuvant (IFA) to achieve a hyperimmune sera panel. The immunization was done for 1, 14 and 28 days with 1 dose of 1 ml antigen containing 106 cfu/ml. The hyperimmunity of sera was confirmed by CFT. B. mallei whole-cell proteome was prepared through sonication and the protein content was visualized by SDS-PAGE and quantified by Western blot using HRP-conjugated rabbit anti-horse IgG. A comprehensive set of positive and negative horse sera validated the test.Three farm horses were subcutaneously immunized with a crude suspension (10B. mallei immunoreactive antigens was seen within the region of 20–90 kDa clearly and the immunoblot was scored positive, while no reaction was seen for the negative sera. The Western blot assay indicated a noticeably higher diagnostic specificity for positive or negative sera of glanders.A ladder pattern of the The whole-cell proteome-based immunoblot proved reliable and straightforward in our study. The prepared antigen was adaptable for application in immunoblotting. We assumed this improved immunoblotting system provides appropriate sensitivity and also specificity expected in serodiagnosis of glanders in endemic areas and typically in less-developed countries. Burkholderia mallei, a Gram-negative bacillus, is the causative agent of the febrile disease known as glanders, a pulmonary disease, and a facultative intracellular pathogen mostly of equidae   and a listed pathogen by Biological Weapons Convention on the grounds of its flexibility to be weaponized  . There icosides) . Glandercosides) . B. mallaponized , 10. In  warfare , 3, 11. B. mallei culture extract (e.g. 0.1 ml) is administrated into the lower eyelid of the suspected equid and reactions are assayed after specific time  was used as a control strain. This non-native strain has been employed for the production line of the mallein at the Razi Vaccine and Serum Research Institute, IR, Iran and was kindly provided by the Swedish cooperative laboratory in 1956. Furthermore, we have isolated 4 new strains of B. mallei  regarding the endemic disease in Iran over a 7-year period ranging from 2010 to 2016. Considering the Biosafety Level 3 (BSL-3) facility, the B. mallei strains were aerobically cultured in nutrient broth supplemented with glycerol 4% for 48 h at 37°C. Whole-cell proteome of bacterial isolates was extracted as follow: concisely, the culture pellet was centrifugation at 9,000 × g for 15 min at 4°C then washed thrice with ice-cold phosphate buffered saline . The pellet was resuspended in PBS containing 1 mM phenylmethane sulfonyl fluoride (PMSF) and sonicated on ice at 90 amplitude for 5 cycles of 1 min pulsing with 1 min rests between the pulses  then centrifuged at 9000 × g for 10 min. The supernatant was treated with 1 ml lysis buffer . The supernatant was precipitated with tricarboxylic acid (TCA) . The washed pellet was resuspended in PBS   of heat-inactivated B. mallei formulated with incomplete Freund’s adjuvant (IFA)  to achieve a hyperimmune sera panel. The immunization was done for 1, 14, 28 days with 1 dose of 1 ml antigen containing 106 cfu/ml. The titer was parallel analyzed with CFT and Western blot assay parallelly using the homologous antigen twice-weekly and for 10 weeks followed by the first step of immunization  coated with 10 μg/ml B. mallei LPS antigen in 0.05 M carbonate-bicarbonate buffer, pH 9.6 at 4°C overnight. The plates were twice washed with PBS-T, pH 7.2  and blocked with 2.5% skimmed milk in PBS-T for 2 h at 37°C. Test sera diluted 1:200 in blocking buffer was appended in duplicate wells for 1 h at 37°C. Then, the wells were washed five times with PBS-T. Rabbit anti-horse (IgG) antibodies conjugated to horseradish peroxidase (HRP) diluted 1:10,000 were added to the wells and incubated for 1 h at 37°C. After washing five times with PBS-T, the plate was developed in the dark for 10 min with TMB  chromogenic substrate. The reaction was stopped by addition of 2.5 M H2SO4 and absorbance was measured at 492 nm in an ELISA plate reader . Cutoff value for indirect ELISA was determined by 15 randomly assigned glanders-negative sera samples according to the above-described method. The cutoff optical density at 492 nm was measured by the addition of 2 standard deviations (SDs) to the mean OD492 of the sera samples, and test samples with an OD492 greater than the cutoff were preserved as positive.A total of 121 sera samples were obtained from different horse populations in various geographical areas of Iran from 2016 to 2018. The sera samples were parallelly monitored by ELISA and CFT for glanders according to the instructions of the OIE Manual of Diagnostic tests and Vaccines for Terrestrial Animals. Appropriate antigen concentration and serum dilution was optimized by checker-board titration method using glandes positive and negative control sera. Indirect ELISA was launched in 96-well microplates   containing 0.1% gelatin. The diluted sera were inactivated for 30 min at 58°C. Twofold dilutions of the sera were prepared using veronal buffer in 96-well round-bottom microtiter plates. Serum, complement, and antigen were gently mixed and incubated for 1 h at 37°C. A 2.5% suspension of sensitized washed sheep red blood cells was added. Plates were incubated for 1 h at 37°C and then centrifuged for 5 min at 600 × g. Serum haemolysis at the 1:5 dilution was considered as negative. The controls of the positive, negative, complement/anti-complement, antigen, and haemolytic system were remarked .B. mallei strains was separated on denaturing 6–12% polyacrylamide gradient gel for 15 h at 25 V according to the Laemmli method. Coomassie brilliant blue G-250 was used for staining and the molecular mass of the proteins was estimated using protein standards.The protein repertoire of B. mallei native strains were equally mixed for further investigation of immunoblotting. A replicate gel was transferred to polyvinylidene difluoride (PVDF) membrane  for 2 h at 300 V. Prior to use, the membrane was activated by soaking in methanol for 20 seconds. The membrane was blocked with 5% BSA for overnight. After being washed with TBS-T  three times, the membrane was cut into slim strips, then incubated with a 1:200 dilution of equine/rabbit sera as the primary antibody, followed by washed three times with TBS-T, and a 1:10,000 dilution of HRP-conjugated rabbit anti-horse IgG as the secondary antibody  in TBS-T for 2 h. The signals were followed by chemiluminescent 3,3′-diaminobenzidine (DAB)  and H2O2 as the substrate.The prepared antigens of the McNemar’s test by the Prism 8.0 statistics tool was used to interpret of the sensitivity, specificity, and positive and negative predictive values of the immune assay methods.B. mallei native strain growth. Regarding the glycerol as the key factor on the growth of B. mallei, once the bacterial strains were cultured in nutrient broth supplemented with glycerol 0.5% and once again increased with glycerol 4%. No difference was seen at protein expression level by SDS-PAGE analysis in both conditions (Two culture conditions were considered for the nditions . The pro492 (mean OD492_2 SDs). One false negative and 2 false positive were found by the indirect ELISA. The sensitivity and specificity of indirect ELISA were evaluated  and , respectively. The positive predictive value (PPV) and negative predictive value (NPV) were estimated  and , respectively. CFT assay indicated that 12 out of 121 sera were positive with sensitivity and specificity of  and , respectively. Only 1 false negative and 1 false positive were found by the CFT. The PPV and NPV of CFT were estimated  and , respectively. Further, sera with suspicious results in indirect ELISA and anticomplementary activity in CFT were retested by Western blotting. 11 ELISA and CFT-positive sera were assayed by Western blot showing ladder-like bands pattern of immunoreactive proteins and were counted positive. Although, 110 out of 121 sera were scored true negative by the Western blot assay. The immunoproteomic strategy using specific immunoreactive proteins of B. mallei was applied for the serodiagnosis of glanders with the high sensitivity and specificity of 100% (95% CI). Furthermore, it seems that the most immunoreactive proteins of B. mallei were almost ranged from 20–90 kDa. The charged strips with the equine sera panel containing immunoreactive protein antigens are shown in P. aeruginosa and B. pseudomallei-mediated hyperimmune sera produced in albino rabbit showed no detectable bands pattern (A total of 13 out of 121 sera samples were positive by indirect ELISA regarding the cutoff OD pattern .B. mallei for serodiagnosis of glanders with a reliable sensitivity and specificity assessment of the immunoreactive proteins. The seroprevalence study was conducted among Iranian horses by using ELISA, CFT and also Western blot with combinations of B. mallei native strains. The ELISA and CFT false positives and distrustful sera were re-tested with Western blot and indicated considerable differences in specificity of serodiagnosis by the Western blot in comparison with the ELISA and CFT. Western blot assay confirmed positive and excluded false positive ELISA and CFT results with a 100% sensitivity and specificity. We know that the diagnosis of glanders is highly problematic due to low awareness of the clinical manifestations of disease, nonexistence of experience among microbiologists outside the endemic areas, lack of suitable culture media and identification systems in the custodian laboratories, and the biosafety facilities (Here, we describe an immunoblotting method based on the whole-cell proteome of B. mallei. Thus, the only successful control option for glanders is testing and isolating positive cases (B. mallei as a hazardous zoonotic pathogen and potential bioweapon agent, little is known about the serodiagnosis of glanders. It is noteworthy that the serological tests for RBPT, IHAT, CIET, and IFAT which are available for diagnosing glanders have also deficiencies and unacceptable specificity and/or sensitivity (B. mallei infection (B. mallei in niches and has formally been prescribed for the international trade of equidae, it has some shortcomings including insufficient sensitivity and specificity, improper results from sera having anticomplementary features as well as false positive/negative results (In an endemic area such as Iran, early detection of the disease would be helpful for controlling glanders by adopting the test and slaughter policy , 7. Currve cases . The effve cases . Despitesitivity . Regardinfection , 19. Alt results , 15, 16. results . CFT may results .B. mallei was made to diagnose positive and negative sera obtained from horses and mules from endemic and non-endemic areas by using the Western blot technique showing a significantly higher diagnostic specificity in comparison with the CFT particularly in glanders-free areas (B. mallei are complicated, time-consuming, and unsafe for the laboratorians (B. mallei and B. pseudomallei have high cross-reactive epitopes in their LPS underlying false positive results in serodiagnosis (B. mallei-specific epitopes as well as genus-specific epitopes.The whole-cell proteome-mediated immunoblotting can be adopted for new diagnostic target antigens existing in the serum. Furthermore, the method is highly specific test for sera with anticomplementary or suspicious reactions in CFT . In a reee areas . Howeveratorians . On the atorians . B. malliagnosis . We assuB. mallei and B. pseudomallei due to the derivation probability of B. mallei from an ancestral B. pseudomallei and having high similarity at their genome level (B. mallei and B. pseudomallei using well-characterized single antigen by recombinant technology or naturally purified are discussed due to cross-reactivity chance (B. mallei and B. pseudomallei. It is important in international trade of animals which have infections with dangerous zoonotic agents B. pseudomallei and/or B. mallei (It is evident that there are difficulties in specific detection and differentiation of me level . The othy chance . Our fin. mallei .B. mallei-infected horses from the neighboring countries can disseminate this disease (Preventing and controlling programs of glanders have been pointed out to health authorities in Iran . The ill disease . Moreove disease . The devIn conclusion, Western blotting is a highly suggestive confirmatory test along with the CFT to increase the serodiagnosis specificity of glanders. So, it can be scheduled as a trustworthy early serodiagnosis test in OIE Manual for eradication programs.


NR1 subunit and thirtyfold decrease of M2 muscarinic receptor transcripts caused by SCT were not counteracted by stimulation, whereas a threefold decrease of AMPAR GluR2 subunit tended to deepen after stimulation. We conclude that LG MNs, supported with proprioceptive stimuli after SCT, do not transcribe the perceived cues into compensatory response at the transcriptional level in the early post-SCT period.Alpha-motoneurons (MNs) innervating ankle extensor muscles show reduced peripheral inputs from Ia proprioceptive afferents and cholinergic afferents after chronic spinalization (SCT). That phenomenon is not observed on ankle flexor MNs, indicating a smaller vulnerability of the latter MNs circuit to SCT. Locomotor training of spinal rats which partially restored those inputs to extensor MNs tended to hyper innervate flexor MNs, disclosing a need for selective approaches. In rats with intact spinal cord 7-days of low-threshold proprioceptive stimulation of the tibial nerve enriched glutamatergic Ia and cholinergic innervation of lateral gastrocnemius (LG) MNs, suggesting usefulness of selective stimulation for restoration of inputs to extensor MNs after SCT. Accordingly, to examine its effectiveness after SCT, tibial nerves and soleus muscles were implanted bilaterally, and for MN identification fluorescence tracers to LG and tibialis anterior (TA) muscles were injected two weeks prior to spinalization. Stimulation of tibial nerve, controlled by H-reflex recorded in the soleus muscle, started on the third post-SCT day and continued for 7 days. Nine days post-SCT the number and volume of glutamatergic Ia and of cholinergic C-boutons on LG MNs was decreased, but stimulation affected neither of them. Postsynaptically, a threefold decrease of NMDAR  Indeed, seven days of continuous bursts of low-threshold stimulation of proprioceptive fibers in the tibial nerve was effective not only in enrichment of glutamatergic Ia but also cholinergic terminals innervating the ankle extensor MNs in rats with intact spinal cord. Immunofluorescence (IF) labeling of vesicular transporter of glutamate, type 1 (VGLUT1), specific for monosynaptic Ia input to MNs  octane , cover-slipped and stored in the dark at 4°C until analysis. The regular staining procedure was preceded by the assay comparing single and triple labeling to test whether simultaneous triple reaction does not impair or cause non-specific antibody binding. Negative controls obtained by omitting the primary antibodies provided no staining.The immunolabeled, retrogradely traced MNs were captured with confocal inverted microscope LSM 780 . Z-stacks of images of labeled MNs and terminals apposing their perikarya consisted of digital slices collected at 0.21 μm intervals with a pixel size of 0.065, using PL APO 40x (1.4 NA) DIC oil-immersion objective. Images were collected at constant exposure parameters for each of four channels detecting Fast Blue or Cholera toxin IF tracers, and IF signal for VGLUT1, VAChT and synaptophysin.The number of examined LG and TA MNs and the number of VGLUT1 IF and VAChT IF boutons abutting on these MNs are shown in the Z-stacks of images were subjected to deconvolution procedure using Huygens Professional software  to reduce the image distortion arising from light scattering. Based on random set of images an experimentally blind rater determined parameters of the deconvolution algorithm to yield maximal resolution for each antibody. The level of background and signal to noise ratio was selected for each channel independently.While the presence of apparent appositions at the LM level does not ensure synaptic contact, we made an assumption of a contiguity of glutamatergic and cholinergic boutons to the cell body or proximal part of a dendrite, when identified with VAChT cytoplasmic staining and verified with bouton overlap with synaptophysin, examined on single optical sections  and analysis of 30–50% of the MN soma surface, done by means of ImagePro Premiere software . Created for our purpose macro was used for semiautomatic measurement of the number and volume of individual IF-synaptic terminals on the surface of MNs.The Z-stack of digital images allowed for 3D reconstruction (voxel size (X/Y/Z) of 0.065 x 0.065 x 0.21 μmTo render a detailed surface of MNs, their 3D masks were created based on IF signal of VAChT detected in the MN cytoplasm and of synaptophysin signal adjacent to the plasma membrane see . The thr2 (mean +/-SD). In order to normalize the results, the density of synaptic boutons was expressed as their number abutted on 3000 μm2 of the lateral surface of the soma, i.e. the same fragment of the MNs soma, which we referred to in our previous study [3) and cholinergic terminals (objects larger than 5 μm3) for the intact animals were adopted from other studies [The mean of the lateral surface of analyzed fragments of the MNs soma was balanced between groups and amounted to 4324 +/- 2018 μm studies –30 and u™ PicoPure™ RNA Isolation Kit, KIT0204, Applied Biosystems) were placed in the tube holder for sample collection. By means of LMD system microscope  attached to the software unit, MNs were identified, selected and then cut with UV laser. The dissected sections of MNs dropped via gravity into prepared tube cap. To limit RNA degradation, samples were collected for up to 60 min per slide. Collected MNs were incubated with extraction buffer for 30 min at 42°C to lyse the cells; lysates were centrifuged at 800 x g for 2 min. Then samples were immediately frozen on dry ice and stored at −80°C.Rats were deeply anaesthetized with a lethal dose of Morbital  and perfused transcardially with ice-cold saline. Lumbar spinal cord L3-6 segments from CN (N = 9), SCT (N = 9), and SCT + electrode-implanted (N = 6) rats were dissected immediately in the cold room, frozen on dry ice and stored at -80°C. The spinal cord segments were surrounded by Jung tissue-freezing medium  and cut into 20 or 25 μm thick longitudinal sections on the cryostat (Leica CM1850) at −20°C. Sections were mounted onto RNase-free PET-Membrane Frame Slides  and immediately placed in dry ice-precooled box. Slides were stored at -80°C until processing . Prior to laser miscrodissection (LMD), sections were dehydrated stepwise in 70%, 80%, 90%, 2x 100%, ethanol for 30s each, submerged in 2x 100% xylene to remove ethanol  and air dried for 3–5 min. Leica Laser Microdissection system LMD7000 was used to isolate LG and TA MNs labeled with neurotracers. Single slides with dehydrated sections were placed on the holder of the LMD system and RNase-free caps of 0.2 ml tubes filled with 20 μl extraction buffer  according to the manufacturer’s protocol, followed by RNA pre-amplification with the use of QuantiTect® Whole Transcriptome kit  to synthesize the cDNA. The final concentration of the cDNA was determined by utilizing Quant-iT™ PicoGreen™ dsDNA Assay Kit . Gene expression level was analyzed by RT-qPCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control gene. The RT-qPCR reactions were performed in a 20 μl reaction mixture containing 7.4 μl PCR grade H20, 10 μl LightCycler® 480 Probes Master solution , 0.4 μl forward primer (20μM) of target gene, 0.4 μl reverse primer (20 μM) of target gene, 0.4 μl probe (10 μM) of target gene, 0.1 μl forward primer (20 μM) of GAPDH gene, 0.1 μl reverse primer (20 μM) of GAPDH gene, 0.2 μl probe (10 μM) of GAPDH gene, 1 μl cDNA (diluted 20 times according to the concentration). Roche LightCycler® 96 Instrument  was used to run the reactions with the following thermal cycling profile: preincubation at 95°C for 10 minutes, 60 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds. Data were analyzed by using the LightCycler software. The 2-ΔΔCt method has been used as a relative quantification strategy for data analysis based on the target and reference genes’ Ct (cycle threshold) value, the cycle number at which the amplification curve reaches the threshold line.The total RNA from LG and TA MNs was isolated by using Arcturus PicoPureTarget-specific probes, forward and reverse primers designed by Universal Probe Library Assay Design Center were used .The Shapiro-Wilk test was used to verify normality of data distribution. The homogeneity of variance in the groups was analyzed with the Leven test. Because normality of data distribution was not met in some experimental groups, the non-parametric Wilcoxon and Mann-Whitney U tests were used for comparisons of related samples and independent samples, respectively. STATISTICA 13 software  was used for the data analysis.Before SCT the control H-reflexes tested with single pulses delivered at 0.3 Hz were observed in all but one animals. The current set at the threshold eliciting M-responses was optimal for eliciting H-reflexes. The latencies of H-reflexes and M-responses in the soleus muscle were approximately 6 ms and 1.5–2 ms, respectively. Two days after SCT we could not elicit the regular H-reflexes to single pulses in four of six animals. However, we observed the recruitment of the complex R2 and R3 responses see  after st2 of the lateral surface of the soma. In CN rats the mean density of VGluT1 IF boutons apposing LG MNs was 11.3 +/- 2.46 (+/- SD) and was non-significantly higher than the density of VGluT1 IF boutons on TA MNs, which amounted to 9.2 +/- 3.26. The mean density of VAChT IF boutons apposing LG and TA MNs was similar, amounting to 12.2 +/- 1.37 and 13.2 +/- 2.02 terminals, respectively.To reveal the effects of SCT on glutamatergic Ia input and cholinergic V0c input to LG MNs, the density and volume of VGLUT1 IF and VAChT IF boutons apposing immunolabeled MNs on the sham-stimulated side of the spinalized rats were compared with those in the non-transected, CN group Figs  and 4. TIn the analysis of the effects of stimulation on these inputs, we compared individual neuroanatomical data with averaged complex R2 and R3 responses collected during the last day of stimulation, to search for the relationship between the degree of input impairment and stimulation effectiveness .The first conclusion emerging from that analysis is a clear differentiation in responses of glutamatergic innervation of LG MNs to SCT. Although in all but one animal the number of VGLUT 1 IF terminals abutting LG MNs decreased after SCT, in half of the group the effect was profound  while in the other half  it did not exceed 25% . In the The second conclusion is that 9 days after SCT the cholinergic innervation of LG MNs was less affected comparing to the group Ia proprioceptive glutamatergic innervation; its loss did not exceed 28% of control values (e.g. in rats 2 and 5). On the stimulated side the number of VAChT IF terminals was slightly higher (rats 2 and 5) or did not change .The effect of SCT on the volume of VGLUT 1 IF boutons was also stronger in the group composed of rats 2, 3, 5 than those of 1, 6, 8 but the changes induced by stimulation were inconsistent in these subgroups. There was a decrease of the volume of VAChT IF boutons after SCT and an inconsistency in their response to stimulation Figs .Seven days of low-threshold stimulation of proprioceptive afferents in the tibial nerve hadn’t change the mean density, neither had it change the mean volume of VGLUT1 IF  and VAChT IF  boutons abutting LG MNs, when compared with the results in the sham-stimulated side .To examine the possibility that the volume of particular subpopulations of terminals was increased by stimulation, a detailed analysis of changes in volume of sized VGLUT1 IF and VAChT boutons on LG MNs was carried out. The analysis showed that both after SCT and stimulation the number of the smallest VGLUT1 IF boutons increased, while the number of the bigger ones decreased. The same pattern of changes was observed in size-distribution of VAChT IF boutons .The effect of SCT on the density of VGLUT1 IF and VAChT IF boutons apposing TA MNs was more pronounced than on LG MNs . The denThe density of VAChT IF boutons was decreased by 34% after spinalization . The mean volume of VAChT IF boutons apposing TA MNs was not affected by SCT .3) occurred  and was not significantly different from a decrease in sham-stimulated hindlimb . A 34% decrease of density of VAChT IF boutons post-SCT  was maintained in the stimulated hindlimb, and did not differ significantly from the decrease, which developed in sham-stimulated hindlimb  subunit, AMPA GluR2  and reference GAPDH gene in microdissected MNs was quantified using duplex real-time RT-PCR. Representative amplification curves for all transcripts are shown in Expression of glutamatergic receptors [NMDA NR1 (Gria2), 25 (Chrm2) and 29 (Grin1), and for the reference gene 18. It indicates that AMPA GluR2 (Gria2) subunit and M2 receptor (Chrm2) transcript levels were of the same order of magnitude, and were approximately 10 times higher from the level of expression of NMDA NR1 (Grin1) subunit (In control samples mean Ct for the target genes was 24 ( subunit . Both glSCT in rats which did not receive electrodes implantation caused a profound decrease of M2 receptor (Chrm2) NMDA and NR1 (Grin1) subunit transcript levels (by 80–90%) both in LG and TA MNs, while AMPA GluR2 (Gria2) subunit transcript change was less (by 20%), but revealed to a similar extent in LG and TA MNs.LG and TA MN expression of AMPA GluR2 subunit was significantly decreased after SCT in implanted rats . LG and TA MN expression of NMDA NR1 subunit tended to decrease ; LG and TA MN expression of M2 receptor was also significantly decreased .Seven days of enhancing direct Ia-signaling to LG MNs did not have significant influence on the expression level of any of the receptors under study neither in LG nor in TA MNs.The aim of the presented experiment was to enhance synaptic Ia proprioceptive input to the MNs innervating the ankle extensor muscles, which were found to be particularly vulnerable to the complete spinal cord transection (SCT) several weeks thereafter , 9. To cIncreased frequency of complex R2 and R3 responses to the burst stimulation indicate, that the H-reflex loop was activated and its effectiveness increased during consecutive days of this stimulation. The continuous bursts were delivered at 40Hz during four 20 min stimulation sessions daily, securing quite intensive activation of low-threshold proprioceptive fibers in the tibial nerve. This stimulation, effective in the intact rats, was primarily addressed to direct glutamatergic innervation of LG MNs by Ia terminals which were identified with VGLUT 1 IF antibody specifically labeling them . Here wend post-surgery day and normalize on the 5th day after SCT [One of the important factors, which could limit the effectiveness of applied stimulation, was its delivery period, largely during the “spinal shock” or rather areflexia period. The duration of the “spinal shock” symptoms, which appear immediately after spinal cord injury, varies depending on the species and severity of the injury . In ratsfter SCT . Both softer SCT . In our fter SCT . The dimfter SCT  and conffter SCT . Moreovefter SCT , and confter SCT , their cAn important factor in these considerations is the transient disappearance of voltage-activated sodium and calcium persistent inward current (PIC) in MNs, which was observed shortly after SCT . The latThis early post-acute period, may not be indicative of how the system will respond at later time points. Some compensatory phenomena, which develop weeks after SCT, may form the basis to extend the application of that demanding approach, used in the current study . Using hGluR2 and NMDA NR1 receptor subunits and M2 muscarinic receptor, similarly in LG and TA MNs. Importantly, our finding on GluR2 loss of transcripts suggests a relative increase of GluR2-lacking, calcium-permeable AMPA receptors (CP-AMPARs) on MNs, that, if confirmed at the protein level, would be in line with reports on that SCI specifically increases postsynaptic localization of CP-AMPARs on them [Our quantitative analysis of glutamatergic Ia and cholinergic inputs to LG and TA MNs carried in this experiment provided evidence, which extends our understanding of the behavior of their preserved inputs and vulnerability of extensor and flexor MNs to SCT. Contrary to selective loss of inputs and dysfunction of extensor MNs developing at 6 weeks after transection , 9, 36,  on them . Functio on them . Periphe on them . ModulatResults collected in this study show, that at the early postlesion period, within a week post-SCT, both groups of MNs respond with loss of inputs and downregulation of their receptors, revealing similar vulnerability to the lesion and suggesting similar impairment of excitatory signaling to them at that postlesion period. Changes, which develop in time and differentiate their state at later period, have the underpinning in their differential functional conditions (unloading of ankle extensor but not flexor muscles) but may also result from differences in their intrinsic properties , 36. OurS1 TableThe occurrence of M1 responses was counted during 3 s time intervals and is presented as a percentage of 118 bursts of stimuli. Counting started at 70, 600 and 1150 seconds of the first training session on days 1 and 7.(DOCX)Click here for additional data file.S1 FigLight green lines indicate the most recent responses; darker green lines indicate superimposed responses to the number of preceding stimuli . A-C. Clear recruitment of H-reflex with small and variable (A) or none  M1.(DOCX)Click here for additional data file.