The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1–RUVBL2–RPAP3–PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins. The R2TP/PFDL co-chaperone facilitates assembly of RNA polymerase II and PI3-kinase-like kinases such as mTOR by a so far unknown mechanism. Here authors provide the cryo-EM structure of human R2TP, which shows how RPAP3 serves as a flexible platform to recruit HSP90 to diverse client proteins. Yeast R2TP complexes comprise four subunits, RuvB-like AAA+ ATPases Rvb1p and Rvb2p, a TPR domain-containing protein Tah1p, and a PIH domain protein Pih1p. Metazoan R2TP complexes contain the orthologous proteins RUVBL1, RUVBL2, RPAP3, and PIH1D1, respectively. However, whereas the TPR domain-containing component of the yeast R2TP complex is a small (12 kDa) protein, Tah1p, in human R2TP this is a large (75 kDa) multi-domain protein, RPAP3 (RNA polymerase II associated protein 3) (or hSPAGH), containing two TPR domains. The C-terminal region in RPAP3 has been annotated as a protein domain (pfam13877), which is also present in other proteins, such as CCDC1038, a dynein arm assembly factor that interacts with RUVBL29.The R2TP/Prefoldin-like (R2TP/PFDL) complex collaborates with the HSP90 molecular chaperone to facilitate assembly, activation, and cellular stability of a range of multiprotein complexes, including RNA polymerase II (Pol II), complexes of PI3 kinase-like kinases (PIKKs) such as TOR and SMG1, and small nuclear ribonuclear protein (snRNPs) complexes, amongst others10. In addition, R2TP/PFDL interacts with additional proteins that serve as adaptors between R2TP/PFDL and the clients (see later)12.In mammals, the R2TP core components associate with additional subunits of the prefoldin (PFDL) module, forming the R2TP/PFDL complex. This PFDL module includes prefoldin and prefoldin-like proteins PFDN2, PFDN6, URI1, UXT, PDRG1, and it associates with two additional components, the RNA polymerase subunit POLR2E/RPB5 and WDR92/Monad13. RPAP3 was then found to be a component of the multi-subunit R2TP/PFDL complex14. Subsequently it was found to associate with Pol II subunits and HSP90 when Pol II assembly is blocked by α-amanitin, implicating both RPAP3 and HSP90 in Pol II assembly in the cytoplasm10. Pol II subunits RPB1, RPB2, and RPB5 all co-precipitate with RPAP3, but RPAP3 seems to associate independently with RPB1 and RPB5-containing complexes, suggesting the existence of different RPAP3 complexes as intermediates in Pol II assembly. RPAP3 also binds some subunits of RNA Pol I and it may therefore play a more general role in the assembly of all RNA polymerases10. The mechanistic details of how RNA Pol II subunits are recruited to R2TP and how R2TP and HSP90 contribute to Pol II assembly are poorly understood. Unconventional prefoldin RPB5 Interactor 1 (URI1) interacts with the RPB5/POLR2E subunit of Pol II, and this suggests that the PFDL module contributes to recruit Pol II assembly intermediates to the R2TP/PFLD complex15.RPAP3 was first identified and named after a systematic analysis of complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry4. This motif is conserved in Tel2p/TELO2, a component of the TTT (Tel2p/TELO2–Tti1p/TTI1–Tti2p/TTI2) complex that also interacts directly with PIKKs, thereby bridging their interaction to R2TP. A similar PIH-binding motif is also found in Mre11p/MRE11A suggesting that R2TP may also play a role in the assembly of MRN complexes involved in DNA double-strand break repair2. Neither Pol II nor snRNPs subunits contain this motif, and must therefore be recruited to R2TP through alternative mechanisms. Biogenesis of box C/D snoRNP requires R2TP and additional factors such as NUFIP1 and the Zinc-finger HIT domain proteins ZNHIT3 and ZNHIT6, which have been proposed to function as adaptors between R2TP and C/D core proteins12. Interestingly, ZNHIT2, another protein of the same family, was recently shown to bind RUVBL2 and regulate assembly of U5 small ribonucleoprotein5. ZNHIT2 may function as a bridging factor between the U5 snRNP and the R2TP/PFDL, a function where the Ecdysoneless (ECD) protein could also contribute16. Human ECD homolog interacts with the pre-mRNA-processing-splicing factor 8 (PRPF8)17, and the R2TP18. Phosphorylated ECD interacts with the PIH1D1 subunit, as well as with RUVBL1 in a phosphorylation-independent manner18. Therefore, it seems that sets of different adaptors collaborate to bring specific clients to R2TP/PFLD.Recruitment of PIKK proteins to R2TP is mediated by the phosphopeptide-binding PIH domain at the N-terminus of Pih1p/PIH1D1, which recognizes a specific phosphorylated acidic motif, generated by casein kinase 2 (CK2)22, while its C-terminal extension couples Tah1p to the CS-domain of Pih1p21. The central region of Pih1p mediates recruitment of Pih1p–Tah1p to the Rvb1p–Rvb2p heterohexameric ring24. The N-terminal PIH domain of Pih1p/PIH1D1 binds a CK2-phosphorylation motif on Tel2p/TELO2, mediating recruitment of the TTT complex to R2TP3. Most recently, we have determined the cryo-EM structure of the intact yeast R2TP complex, in which a single Tah1p–Pih1p sub-complex binds a heterohexameric Rvb1–Rvb2 ring24, a finding subsequently confirmed by others25. In metazoan R2TP, the small (12 kDa) single-TPR domain protein Tah1p is replaced by the much larger (75 kDa) RPAP3/hSpagh whose N-terminal half contains a tandem pair of TPR domains that bind in concert to a single HSP90 dimer2. However, the function of the rest of RPAP3 is unknown. To our knowledge, the subunit stoichiometry and the structural organization of a metazoan R2TP complex have not been determined.Previous structural and biochemical studies have defined most of the pairwise interactions of R2TP core components. The TPR domain of yeast Tah1p mediates interaction with the conserved MEEVD C-terminal tail peptide of HSP90To gain further insight into how R2TP/PFDL functions in the assembly, activation and stabilization of its ‘client’ systems, we have determined the cryo-EM structure of human R2TP core complex. Our data reveal a substantially elaborated architecture compared with the yeast system, in which RPAP3 rather than PIH1D1 plays the central organizational role, incorporating additional domains and functions to address the assembly of a variety of large complexes. We identify the C-terminal domain in RPAP3 as a helical bundle that binds selectively to the ATPase domain of RUVBL2. As well as scaffolding the interaction of PIH1D1 with the RUVBL1–RUVBL2 ring, RPAP3 provides a flexible tether for HSP90, allowing it to interact with a highly diverse set of client proteins and complexes.26. We found that human PIH1D1 protein was also unstable when expressed in isolation, and we used this property to identify a minimal PIH1D1 binding motif in RPAP3 by co-expressing PIH1D1 with GST-tagged RPAP3 constructs and looking for co-purification of PIH1D1 in GST pull-downs from cell lysates. As well as in the full-length GST-RPAP3, we found that constructs that contained residues 400–420 of RPAP3, immediately downstream of the second RPAP3 TPR domain, were able to form a stable and soluble complex with full-length PIH1D1 or its isolated CS domain, when co-expressed .Together, our interaction mapping reveals a very different assembly of the PIH domain and TPR domain-containing components of human R2TP than in the yeast system28. In all cases, we used Adenosine 5′-diphosphate (ADP) in the buffer, which stabilizes RUVBL1–RUVBL2-containing complexes30. When RUVBL1–RUVBL2 was incubated with a fragment of RPAP3 comprising residues 430–665, up to 3 RBDs decorated the ATPase face of the RUVBL ring, one per each RUVBL2 molecule in the complex  bound to PIH1D1, but from which the TPR domains and N-terminus of RPAP3 have been removed (RPAP3-ΔNT) Fig. . These iR2TP images were classified according to the number of RBDs per ring, by masking out the rest of the molecule and without assuming symmetry . Three-dimensional (3D) classification generated subgroups corresponding to small differences in some of the α-helices of the RBD, and the one that reached the best resolution was refined locally to an estimated average resolution of 3.6 Å  Fig. . When th33, INO8011, or yeast R2TP25 are mediated by the face of the ring presenting the DII ‘insertion’ domain. Interaction with the opposite ATPase domain face of the RUVBL1–RUVBL2, as seen here with RPAP3-RBD, has not previously been found, although contacts between this face of the RUVBL ring have been observed in crystals of Rvb1/Rvb2 dodecameric complexes34.Previously described interactions with RUVBL1–RUVBL2 such as in SWR135, which predicted that residues 541–665 comprise 8 highly conserved α-helices preceded by a long and poorly structured region  , and they make contacts with RUVBL2 through H1, H6, and the loop connecting H5 and H6 Fig. . Althoug29 Fig. , suggest29 Fig. .Fig. 6StXenopus  Fig. . In this24  Fig. , in a si24 Fig. . As in t37. HSP90s ability to chaperone this very broad protein clientele is provided by co-chaperones38, which act as adaptors, facilitating recruitment of client proteins to HSP90.HSP90 is required for the stabilization, activation, and assembly of a diverse range of proteins and complexes involved in cellular processes as fundamental and as varied as transcription, cell cycle progression, centrosome duplication, telomere maintenance, siRNA-mediated gene silencing, apoptosis, mitotic signal transduction, innate immunity, and targeted protein degradation40, although this list is likely to be far from complete. It recruits HSP90 via its TPR domain component Tah1p or RPAP3, and at least some of its clientele through a CK2 phosphorylation-dependent interaction with the PIH domain of Pih1p/PIH1D13. The best characterized of these interactions involves Tel2p/TELO2, itself a component of an additional ‘adaptor’ layer, the TTT complex , which ultimately bridges R2TP, and thereby HSP90, to PIKK proteins42. Comparable PIH-binding motifs can be identified in a range of other proteins in yeast and metazoa, suggesting that there are many R2TP-mediated HSP90 dependencies yet to be described, some of which will likely also involve multiple adaptor systems.R2TP/PFDL is the most complex HSP90 co-chaperone yet described, and is known to facilitate HSP90s participation in the assembly of RNA polymerases, PIKK complexes, and snoRNPsThe structure of the human R2TP core components revealed here is well suited to deal with a high degree of diversity in the client proteins it brings to HSP90 Fig. . In both25. Conformational adaptability in the yeast system is thus limited to the inherent flexibility of the ~30 residues linking the last globular domain of the bound HSP90 to the TPR-binding MEEVD motif at its extreme C-terminus.In the yeast system, the single TPR domain of Tah1p is part of a well-ordered cluster of domains along with the CS and PIH domains of Pih1p, that lies in the cup formed by the DII domains of the RUVBL ringThe much more elaborate structure of RPAP3 in human R2TP allows for a far greater level of adaptability, while retaining the topological proximity of TPR and PIH domains required to bring HSP90 and client together. RPAP3 is divided into two regions that are located at the two opposite faces of the RUVBL1–RUVBL2 ring. As revealed in our cryo-EM structures, the interaction of the RBD of RPAP3 with the ATPase face of the RUVBL1–RUVBL2 ring provides a tight anchor for the C-terminus of the protein, while allowing considerable flexibility for the CS-binding segment, TPR domains and N-terminus of the protein on the other face, coupled to the RBD by the long flexible central segment that spans the rim of the ring.25. The RPAP3–PIH1D1 sub-complex behaved as an elongated heterodimer in sedimentation velocity experiment, with an estimated average molecular mass of 103,900 ± 320 Da determined by sedimentation equilibrium assays  into proximity, and at least for yeast suggest some involvement of the ATPase activity of the RUVBL proteins in modulating this, although the significance of this for R2TP function in vivo is far from clear. The ultimate question of how HSP90 functions with R2TP/PFLD to facilitate the assembly of large multiprotein complexes such as Pol II remains to be determined.Along with the structure of the yeast R2TP complexRuvBL1 and untagged RuvBL2 were cloned as indicated in Lopez-Perrote et al.28. For pull-down experiments, a 3xMyc tag was incorporated to the N-terminus of RUVBL1. The RPAP3 full length (FL) was purchased from GenScript. The RPAP31-430, RPAP31-400, RPA31-420, RPAP3395-665, RPAP3430-665, RPAP3523-665, RPAP3430-541, RPAP3541-665, and RPAP3395-665 (RPAP3-ΔNT) gene truncations were cloned using NdeI sites and ligation-free cloning using infusion cloning (Clonetech lab Inc.) into a modified pGex6p modified plasmid named p3E , which resulted in N-terminal GST-tagged RPAP3 genes  and RuvBL2 gene was cloned into pET28b using NdeI and BamHI sites. Human Hsp90 full-length beta gene was cloned into modified pET28b plasmid (contacting 6His-2Xstrep-tags) using NdeI, which resulted in 6His-2Xstrep-PreSc-Hsp90FL beta gene.The human Rosetta (DE3) pLysS cells (F−ompT hsdSB(rB− mB−) gal dcm (DE3) pLysSRARE (CamR), Merck Millipore Ltd.). The cells were grown in the presence of ampicillin and kanamycin antibiotics at 37 °C until the cells reached their log-phase. Then the cells were induced by the addition of 1 mM IPTG. The cells were further grown at 25 °C overnight for protein expression. The cell mass was pelleted by spinning the cell culture at 6238×g for 10 min. The cells were lysed using a sonicator in 20 mM HEPES pH 7.5, 140 mM NaCl (HEPES buffer) and 1 tablet of EDTA-free protease inhibitor (Sigma-Aldrich Ltd.). The cell lysate was centrifuged at 20,000×g for an hour at 4 °C. The clear supernatant was loaded on to the equilibrated Talon beads in 20 mM HEPES pH 7.5, 140 mM NaCl for His-tag affinity-chromatography. The beads were washed with HEPES buffer to remove the contaminant proteins. The proteins of interest were eluted with 500 mM imidazole in HEPES buffer. The eluted proteins were analyzed by 4–12% SDS-PAGE and concentrated to 6 mg ml−1 using viva-spin . The proteins were further purified by size-exclusion chromatography (SEC) using S200 10/300 column . The GST-tagged RPAP3FL and RPAP3 shorter gene constructs alone and in complex with PIH1D1 full length and PIH1D1180-290 were expressed in Rosetta (DE3) pLysS cells. The cells were lysed similar to RUVBL1/2 and HSP90 beta. The proteins were purified using GST-tag affinity chromatography by adding the clear cell lysate to the GST-beads. The GST-bound proteins were washed with HEPES buffer and they were eluted with 50 mM glutathione. The proteins were incubated with PreScission protease (3C protease) to cleave the GST-tag at 4 °C overnight and the proteins free of GST-tag were concentrated to 20 mg ml−1 using viva-spin with MWCO of 10k. The proteins were further purified by gel filtration chromatography using S200 26/60 column in degassed 20 mM HEPES pH 7.5, 500 mM NaCl. For those pull-down experiments using untagged RUVBL1, 6xHis-tagged RUVBL1 was purified and the tag cleaved using PreScission protease.The human RUVBL1–RUVBL2 protein complex and RUVBL2 used in the pull-down experiments, and human HSP90 beta were transformed into 28. N-terminal His10-tagged human RuvBL1 and untagged human RuvBL2 were co-transformed into Escherichiacoli BL21 (DE3) cells grown in LB medium. The lysate was applied to a HisTrap HP column  equilibrated in 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 10% (v/v) glycerol, and 20 mM imidazole. Elution was performed using a 20–500 mM imidazole gradient, followed by a preparative SEC using a Sephacryl S300 column  equilibrated in 50 mM Tris, 300 mM NaCl and 1 mM DTT. The uncropped SDS-PAGE gels of the SEC of the RUVBL1–RUVBL2 and RPAP3–PIH1D1 used for cryo-EM are shown in Supplementary Fig. RUVBL1–RUVBL2 complexes used for cryo-EM studies were produced and purified as before180–290 complexes were mixed with 30 μl GST beads, which were equilibrated in 50 mM HEPES pH 7.5, 140 mM NaCl. Sixty micromolar of the RUVBL2 alone and RUVBL1–RUVBL2 complex were added to the above mixture for the interaction mapping study. In these experiments, a 3xMyc tag was incorporated to the N-terminus of RUVBL1 to help in distinguishing RUVBL1 and RUVBL2 in SDS-PAGE. The above reaction mixture was incubated for 45 min at 4 °C rotating at 20 rpm min−1. The beads were washed three times with 500 μl of HEPES buffer and the bound fraction was eluted with 50 mM glutathione.Twenty micromolar GST-RPAP3 (full length and different lengths of RPAP3) and RPAP3–PIH1D1 and RPAP3–PIH1D1541–665-RuvBL2FL and Gst-RPAP3541–665-RuvBL2 ∆DII. The interactions were analyzed using GST-affinity chromatography and the bound fractions were eluted with 50 mM glutathione in the 20 mM HEPES pH 7.85, 140 mM NaCl, and the quality of the protein complex was visualized using SDS-PAGE.Human R2TP complex assembly was monitored by pull-down. For this, the purified RPAP3–PIH1D1 and RUVBL1–RUVBL2 proteins were used for the R2TP complex assembly. The 20 μM GST–RPAP3–PIH1D1 complex and 60 μM RUVBL1–RUVBL2 proteins were used in the experiments; 30 μl GST-beads  equilibrated in 50 mM HEPES pH 7.5, 140 mM NaCl (HEPES buffer) were added to the above protein complexes. Then the protein mixture was incubated for 45 min rotating at 20 rpm at 4 °C. The beads were washed three times with HEPES buffer to remove the non-specific proteins bound on to the GST-beads. The bound fraction was eluted with 50 mM glutathione in 20 mM HEPES, pH 7.5, 140 mM NaCl. For the co-expression experiments shown in Fig. To image R2TP at high resolution, 0.45 μM RUVBL1–RUVBL2 (estimated as dodecamers) was incubated with 9 μM of RPAP3–PIH1D1 complex for 20 min in ice and the mix dialyzed for 5 h against 25 mM HEPES pH 7.8, 130 mM NaCl, 10 mM 2-Mercaptoethanol. After dialysis, the sample was recovered and incubated with ADP (pH 7.0) for 1 h at a final concentration of 0.5 mM. Subsequently, aliquots of 2.5 μl were applied to Quantifoil R1.2/1.3 carbon grids after glow-discharge, and then flash frozen in liquid ethane. An initial test data set was obtained in Grenoble Instruct Center, France, using a FEI Polara microscope operated at 300 kV. High-resolution structures were obtained from data collected in a Titan Krios , automatically, with three images per hole, using a GATAN K2-Summit detector in counting mode and a slit width of 20 eV on a GIF-Quantum energy filter , with dose weighting. A manually picked subset of micrographs was used to obtain 2D references for template-based particle picking. The selected particles were then submitted to several rounds of 2D and 3D classifications, to discard low-quality particles and some remaining RUVBL1–RUVBL2 dodecamers. Low-pass filtered versions of previous structures were used as starting point of classifications and refinements, to reduce bias.As general methodology, MotionCorr245.A specific classification protocol was designed to analyze the stoichiometry of RBDs bound to the RUVBL ring. For this, Class3D in RELION was used but using a mask covering the edge of the RUVBL ring and the regions outside the ring. Particles were split in up to eight groups using this focused classification strategy. The majority of particles were grouped into one class, containing 3 RBDs and corresponding to 67.3% of the particles. The remaining classes corresponded to particles containing 2 RBDs (16.4%) or 1 RBD (16.3%). Images of R2TP containing 3 RBDs were then processed applying 3-fold rotational symmetry and using standard procedures in RELION32. For this, each particle was rotated 120 and 240° so that all RBDs were then placed in the same position. Subsequently, particles were classified using the Class3D utility in RELION and using a mask representing 1 RBD and the ring of RUVBL1–RUVBL2. The most populated class was automatically refined using the Ref3D utility using the same mask used for Class3D, local search of angles and without applying symmetry. Further details on the strategy applied here can be found in the Methods section of Zhou et al.32. When applied to our data, resolution was improved from 3.8 Å when applying 3-fold rotational symmetry to 3.57 Å, estimated using gold standard Fourier Shell Correlation (FSC) between two independent maps using cut-off of FSC = 0.143. B factor sharpening was performed using automatic procedures in Relion2. Local resolution was estimated using ResMap46. Structures were visualized using UCSF Chimera47.For the refinement of the RUVBL1–RUVBL2–RBD complex at high resolution, 96,406 particles showing the best parameters after 2D classification in RELION were selected and subjected to a round of automatic 3D refinement in RELION to generate a consensus 3D model. When refinement was performed without applying rotational symmetry, similar results were obtained, but some differences in the quality of the different RBDs bound to one RUVBL ring, suggested that the complex had a rigid conformation at the RUVBL ring, and relatively flexible RBDs . To improve the quality of the structure defining the interaction between the RBD and the RUVBL ring, we applied the method previously developed to solve a similar situation for the structure of the apoptosome430–665, and the reconstitution of R2TP using the RPAP3–ΔNT–PIH1D1 sub-complex instead of RPAP3–PIH1D1 were performed with the same protocol used for the R2TP used in cryo-EM experiments. For consistency, all these observations were performed with the same buffer conditions used for the assembly of R2TP, and in the presence of ADP. Vitrifications, the general image processing, 2D classifications, and the generation of 2D averages and 3D volumes were performed following similar strategies to those described for the R2TP images, but the cryo-EM micrographs were collected in a 200-kV FEI Talos Arctica operated with a FEI Falcon II detector and located at the Centro Nacional de Biotecnologia (CNB) in Madrid.The reconstitution of complexes between RUVBL1–RUVBL2 and RPAP3For refinement of the flexible regions of the R2TP complex, the same set of 96,406 particles used to resolve the structure of RUVBL1–RUVBL2–RBD was classified searching for a subset of particles of R2TP with a more homogenous conformation for the flexible regions after extensive 3D classification steps using Relion. This subset, containing a selection of 27,385 particles, was then refined, converging to a structure with an estimated average resolution of 8.72 Å using gold standard Fourier Shell and FSC = 0.143.To analyze the structural details for the interaction between PIH1D1 and RUVBL1–RUVBL2, the images of R2TP complexes containing 3 RBDs were refined without symmetry and using a mask that removed the influence of the flexible regions except the vicinities of the DII domains in RUVBL1–RUVBL2. One subset of 182,351 particles corresponding to complexes containing 3 RBDs and that displayed a defined density in DII-domain face of the RUVBL1–RUVBL2 reached an estimated resolution of 6.58 Å using gold standard Fourier Shell and FSC = 0.143.35, which provided up to five different atomic models for the query sequence. The best solution according to the I-TASSER scoring was fitted within the target cryo-EM map.De novo modeling of the C-terminal domain of RPAP3 was performed using a strategy based on homology modeling and molecular dynamics simulation. First, analysis of the sequence in the secondary structure prediction server PSIPRED revealed 8 α-helices consecutively, starting from Ala547 to C-terminus Gly665. A long and disordered region of RPAP3 N-terminal to this helical domain contributed to a clear identification of the domain. The sequence of RPAP3-RBD domain (residues 541–665) was submitted to I-TASSER homology modeling server48  simulations in AMBER29 into the cryo-EM map with the addition of the model generated for the RBD using AMBER. The full structure of the RUVBL1–RUVBL2–RBD was refined using Phenix49 and Coot36.A model for RUVBL1–RUVBL2–RBD was built initially by the fitting as a rigid body of the crystal structure of the human RUVBL1–RUVBL2 truncated hexamer (PDB 2XSZ)The information of the crystal structure of the RPAP3 fragment comprising residues 578–624 helped to model the connectivity between helix 5 and helix 6 of the RBD, which faces RUVBL2. Density for side chains was also visible in most of the helices of the RBD  was used to analyze the fractions.For the experiments using GraFixN-hydroxysuccinimide (NHS) esters disuccidinimidyl suberate (DSS H12/D12) and bis-sulfosuccidinimidyl suberate (BS3 H12/D12)  at a final excess concentrations of 100 and 250×. The reactions were incubated for 45 min at 37 °C, and quenched by adding 50 mM NH4HCO3  for another 15 min.R2TP was reconstituted as for the cryo-EM experiments and cross-linked with isotopically coded 4HCO3 to reach 1 mg ml−1 final protein concentration. The sample was then reduced using 10 mM DTT and alkylated with 50 mM iodoacetamide. Subsequently, proteins were digested with trypsin  using 1:20 enzyme-to-substrate ratio, at 37 °C, and the incubation was done overnight. A final concentration of 2% (v/v) formic acid was added to acidify the samples, and the peptides were fractionated by peptide SEC in a Superdex Peptide 3.2/300 column  with 30% (v/v) acetonitrile/0.1% (v/v) TFA as mobile phase and using a flow rate of 50 μl min−1. Fractions were collected, lyophilized, and resuspended in 2% (v/v) acetonitrile and 2% (v/v) formic acid.The cross-linked sample was freeze-dried and then resuspended in 50 mM NH−1. Peptides were separated on a C18 Acclaim PepMap100 3 μm, 75 μm × 250 mm nanoViper  and eluted with a acetonitrile gradient. The analytical column outlet was directly interfaced via a nano-flow electrospray ionization source, with a hybrid dual pressure linear ion trap mass spectrometer . A resolution of 30,000 was used for data-dependent analysis for the full mass spectrometry spectrum, followed by 10 MS/MS spectra in the linear ion trap. Mass spectrometry spectra were collected over a 300–2000 m/z range. MS/MS scans were collected using threshold energy of 35 for collision-induced dissociation.Fractions were analyzed by nano-scale capillary LC–MS/MS using an Ultimate U3000 HPLC  and a flow of approximately 300 nl minhttp://prottools.ethz.ch/orinner/public/htdocs/xquest/). The following criteria were used for the selection of cross-linked precursor MS/MS data: a mass shift of 12.07532 Da among the heavy and the light cross-linkers; precursor charge ranging from 3+ to 8+; maximum retention time difference 2.5 min. Searches were performed against an ad hoc database containing the RUVBL1, RUVBL2, PIH1D1, and RPAP3 sequences plus their reverse as decoy. A number of parameters were set to perform the xQuest searches: maximum number of missed cleavages (excluding the cross-linking site) 3; peptide length 4–50 amino acids; fixed modifications carbamidomethyl-Cys (mass shift 57.02146 Da); mass shift of the light cross-linker 138.06808 Da; mass shift of mono-links 156.0786 and 155.0964 Da; MS1 tolerance 10 ppm, MS2 tolerance 0.2 Da for common ions and 0.3 for cross-link ions; search in enumeration mode (exhaustive search). The following criteria were used to filter search results: MS1 mass tolerance window −3 to 7 ppm. Finally, each MS/MS spectra was manually inspected and validated.For data analysis, Xcalibur raw files were converted into the open mzXML format through MSConvert (Proteowizard) with a 32-bit precision, and the converted files were directly used as input for xQuest searches on a local installation . This was equipped with UV-VIS absorbance and Raleigh interference detection systems, and the sedimentation profiles were recorded at 280 nm. Least-squares boundary modeling of sedimentation velocity data and the continuous distribution c(s) Lamm equation model was used to calculate sedimentation coefficient distributions, as implemented by SEDFIT 14.151. The program SEDNTERP52 was used to correct experimental s values to standard conditions  to obtain the corresponding standard s values .Four-hundred microliters of 10 μM and 5 μM RPAP3–PIH1D1 prepared in 25 mM Hepes, 130 mM NaCl, 0.1 mM TCEP, pH 7.8, were loaded into analytical ultracentrifugation cells. The experiments were carried out at 10 °C and 149,103×g to 6532×g and at 286 nm, and using the same experimental conditions as those described for the Sedimentation Velocity experiments. A high-speed centrifugation run using 185,795×g was performed to estimate the corresponding baseline offsets after the last equilibrium scan. Weight-average buoyant molecular weights of proteins were determined by fitting a single species model to the experimental data using the HeteroAnalysis program, and corrected for solvent composition and temperature using the program SEDNTERP52.Short column (90 μl) Sedimentation Equilibrium experiments were carried out at speeds ranging from 4536×523–665 fragment was cloned into the NdeI and BamHI site of the p3E plasmid (home grown plasmid from University of Sussex). BL21 (DE3) E. coli cells  λ(DE3 *lacI lacUV5-T7gene 1 ind1 sam7 nin5), NZYTech) were transformed with p3E plasmid containing RPAP3523–665. The transformed cells were grown in LB media at 37 °C for 5 h followed by induction with 1 mM IPTG. The cells were further grown for 15 h at 20 °C. Cells were harvested using 5000×g for 10 min. The cell pellet was re-suspended in 20 mM HEPES pH 7.8, 140 mM NaCl, 0.5 mM TCEP, and sonicated at 4 °C. The cell lysate was spun at 20,000 g for 1 h at 4 °C and the supernatant was used for the GST affinity chromatography. The purified GST-RPAP3523–665 protein was treated with PreScission protease overnight at 4 °C to remove the GST-tag and the protein was further purified by SEC.The gene encoding RPAP3523–665 protein was concentrated to 8 mg ml−1 using 10k MWCO viva-spin (Sartorius). The crystallization trials were set up using 0.2 μl protein and 0.2 μl crystallization screen buffer using sitting drop method. The crystallization trial trays were incubated at 14 °C. Small crystals appeared in 0.5 M potassium thiocyanate, 0.1 M bis-tris propane pH 7.0 (well H6) of SaltRX crystal screen (Hampton research Ltd) after 1 month of incubation. The crystals were flash frozen in liquid nitrogen using 30% glycerol as a cryo-protectant and the data were collected at Diamond Light source, UK. Wavelength used was 0.9763 Å.Purified RPAP353, Xia254, REFMAC55, BUSTER (https://sbgrid.org/software/titles/buster), and COOT36, together with the ARCIMBOLDO software56 ; EMD-4287 (R2TP-1RBD); EMD-4290 (R2TP-subgroup1); EMD-4291 (R2TP-subgroup2); PDB 6FO1 (model of RUVBL1–RUVBL2–RBD with 1RBD), and PDB 6FM8 .Suplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Movie 1


Their physicochemical properties were examined by field-emission scanning electron microscopy, X-ray diffraction analysis, Fourier-transform infrared spectroscopy, and UV-visible absorption spectroscopy. It was shown that their performances were highly dependent on the morphology, thermal treatment procedure, and TiO2 overlayer coating.Performing diverse application tests on synthesized metal oxides is critical for identifying suitable application areas based on the material performances. In the present study, Ni-oxide@TiO To syntpability . Kim et  [2 NTAs . The enh2 to NiO . Yu et atability . Disinfesite NPs . Chockalf phenol . Howeverlue (MB) . Shinde  defects . Ong et ·g−1·h−1 . Gao et gnostics ,29,30,312 and TiO2@NiO core-shell materials were reported, until now, thorough studies on the diverse applications of the same materials treated with different methods have not been reported. The novelty of the present study stems from the use of NiO@TiO2 core-shells in three different application areas—i.e., photocatalytic dye degradation for water treatment, CO oxidation for air treatment, and supercapacitors for energy storage. In addition, the application efficiency was found to be affected by the thermal treatment procedure. The results obtained here can be very useful for the development of hybrid materials in various fields.Although NiO@TiOx) and B (NiOy) samples resembled hexagonal plates and short rods, respectively, while their color appeared bluish-green, which is characteristic of Ni(II) complexes [x-500 °C) and B1 (NiOy-500 °C)—the color changed to grey, indicating a modification in crystal structure. In addition, the morphologies underwent some changes, exhibiting particles of various sizes and worm-like structures of 1–2 μm, respectively. In the case of the A2 (NiOx@TiO2-500 °C) and B2 (NiOy@TiO2-500 °C) samples, obtained by coating with TiO2 and subsequently annealing (500 °C) the A and B samples, the colors changed to dark yellow. The particle sizes (100–300 nm) of sample A2 were bigger than those of sample A1. Sample B2 showed an aggregated sphere-like morphology, with sizes of 100–400 nm. The samples obtained upon TiO2 coating and annealing of the A1 and B1 samples are denoted as A3 (NiOx500 °C@TiO2-500 °C) and B3 (NiOy500 °C@TiO2-500 °C). The A3 and B3 samples had a similar yellowish-grey color as well as a sphere-like morphology with some small particles.Scanning electron microscopy (SEM) analysis was conducted to examine the sample morphology . The moromplexes ,33. Hexa2 (reference code: 98-016-1897) [x-500 °C) and B1 (NiOy-500 °C) samples had identical XRD patterns matching that of cubic NiO (reference code: 98-064-6098). The three major peaks at 2θ = 37.3°, 43.0°, and 62.9° can be assigned to the (111), (002), and (022) planes of the cubic crystal phase, respectively. The different Ni complexes of Ni hydroxide and oxalate assumed the same NiO structure upon thermal annealing at 500 °C. Cui et al. synthesized Ni(OH)2 nanosheets and flower-like microspheres by hydrothermal method, using ammonia and polyvinylpyrrolidone at 150 °C for 15 h, and obtained NiO nanosheets and NiO microspheres by thermally annealing the corresponding samples at 500 °C [2-coated samples , XRD patterns corresponding to that of tetragonal anatase TiO2 (reference code: 98-020-2243) were observed. The strongest peak at 2θ = 25.3° for TiO2 corresponds to the (011) plane of the tetragonal phase. For samples A2 (NiOx@TiO2-500 °C) and B2 (NiOy@TiO2-500 °C), additional XRD peaks were observed and attributed to hexagonal Ni(II) titanate NiTiO3 (reference code: 98-006-6198). Kim et al. also reported the formation of the NiTiO3 phase for thermally annealed NiO-TiO2 NTAs at 600 °C [3 phase is commonly formed by heating NiO and TiO2 via NiO + TiO2 + heat → NiTiO3. Based on the above results, the A2 and B2 samples were NiO@TiO2 core-shell structures with interfacial NiTiO3, whereas the A3 and B3 samples were NiO@TiO2 core-shell structures with no XRD detectable interface species.16-1897) ,35,36,37t 500 °C . For the−1 for the A and B samples, respectively [−1 [2-coated samples, broad peaks were commonly observed at ~650 cm−1, and attributed to the Ti–O–Ti vibration of the TiO2 lattice [ectively ,38. For vely [−1 ,38. For  lattice .x) and B (NiOy), two strong absorption regions were observed at ~400 and ~700 nm, attributed to the d-orbital bonding transitions commonly observed for Ni(II) complexes [3A2g to the excited states of 3T2g and 3T1g) of Ni(II) with octahedral coordination [2 core-shell structures , band gap absorption edges were observed near 500 nm.Ultraviolet-visible (UV-Vis) absorption spectra  were obtdination . For the2 is widely used in photocatalysis, the NiO@TiO2 core-shells  were tested for photocatalytic dye degradation [3/TiO2 could govern the surface interactions between MO and the B2 sample surface. The order of dye adsorption was found to be MO < RhB < MB for the A2, A3, and B3 samples, and RhB < MO < MB for the B2 sample. The B2 sample showed the best adsorption performance. More interesting results were observed by increasing the visible-light exposure time. Among the three dyes, MO was the fastest to degrade, showing a similar behavior on all the samples. The degradation rate typically showed the following order: RhB < MB << MO. In addition, MB exhibited similar degradation in all the samples; conversely, RhB showed drastic differences between the different samples. The photocatalytic activity for RhB showed an order of B3 << A3 < A2 < B2. The B3 sample showed negligible photocatalytic activity for RhB, while the B2 sample showed a good catalytic activity, comparable to that of MB. The A2 sample showed better activity than the A3 sample for photocatalytic RhB degradation. It appeared that the interfacial NiTiO3 phase formation (for A2 and B2) facilitated the RhB degradation. For all three dyes, the B2 sample showed the best catalytic performance for mixed dye degradation. Some studies on catalysts for the photocatalytic degradation of mixed dyes are summarized in As TiOradation ,49,50,51radation ,43,44,45radation ,43,44,452. The electron (e−) and hole (h+) pairs are separated by interfacial charge transfer at the interface of the p-n junction [2; they are then captured by oxygen to generate active •O2− (or •O−), which can further react with H+ to form •O2H (or •OH). Consequently, the active species of •O2−, h+, and •OH are used for dye degradation. The summarized mechanism is written below [As the dyes and samples absorb visible light, photogenerated electron hole pairs can be formed by direct visible-light absorption by mixed dye and NiO@TiOjunction ,6,18,46.en below ,49,50,51Photocatalytic mixed dye degradation under UV light (365 nm) was also tested . Unlike 2 (mass = 44 amu) production by CO oxidation was monitored with the increasing reaction temperature by using mass spectrometry [x) and B (NiOy)—the CO2 production onsets were observed at 265 and 300 °C, respectively; while, in the second runs, they were observed at 300 and 250 °C, respectively. In the second run, sample A showed degraded activity (by +35 °C), whereas sample B showed enhanced activity (by −50 °C). The Ni hydroxide (sample A) and Ni oxalate (sample B) complexes were found to change to the same NiO crystal phase after the CO oxidation reactions, based on the XRD results. For sample A1, the onsets were observed above 470 °C in the first and second CO oxidation runs. The catalytic activity dramatically degraded for sample A1 with NiO crystal phase. For sample B1, the onsets were observed at 300 and 350 °C in the first and second runs, respectively. Although the B1 sample showed the same NiO crystal phase, its catalytic activity was considerably superior to that of sample A1. Similarly, samples A2 and B2 showed an onset above 450 °C and at 300 °C, respectively. Samples A3 and B3 showed similar onsets near 400 °C for the first and second runs. As a result, the sample preparation method is crucial to determine the catalytic activity. In the present study, the TiO2 coating on the B2 sample did not to degrade the CO oxidation activity. However, when Ni(OH)2 plates were used as starting material, the CO oxidation activity significantly decreased.The materials were tested as CO oxidation catalysts. trometry ,54,55,56− → NiOOH + e− + H2O [2-coated samples (A2 and A3) were found to be identical, 50 F/g. For sample B1 with NiO crystal phase, the specific capacitance was measured to be 189 F/g, lower than that of the A1 sample (320 F/g). However, the other two TiO2 coated samples, B2 and B3, exhibited values of 211 F/g and 134 F/g, respectively, which were higher than those of the A1 and A2 samples (50 F/g). Kim et al. reported specific capacitances in the range of 40–100 F/g for NiO-TiO2 NTAs and 120–300 F/g for NiO-TiO2 nanotube films [2–NiO hybrid materials are summarized in 2 was mainly core [2 core-shell structures has not been reported. The high-frequency region of the impedance Nyquist plots with real (Z′) and imaginary (Z′′) parts are shown in Furthermore, supercapacitor performance tests were briefly performed. The related results of cyclic voltammograms (CVs), galvanostatic charge/discharge curves (CD), and impedance plots are shown in e− + H2O ,57. The e− + H2O ,22,57. Tnly core ,59. To tx (A) and NiOy (B)—were synthesized by hydrothermal method as described below, and then used as starting materials. For material A, 0.475 g of NiCl2·6H2O  was dissolved in 40 mL of deionized water, and 2 mL of 1.0 M NaOH solution was added to it. The solution was then transferred to a Teflon-lined stainless autoclave and placed in an oven setting at 120 °C for 12 h. For material B (NiOy), 0.951 g of NiCl2·6H2O was dissolved in a mixed solvent (20 mL of H2O + 20 mL of ethylene glycol); then, 1.0 mL of 0.1 M NaOH solution and 0.238 g of oxalic acid  were added to it. Upon complete dissolution, the entire solution was transferred to a Teflon-lined stainless autoclave and placed in an oven setting at 120 °C for 12 h. After the hydrothermal reaction, the obtained precipitates were fully washed with deionized water and ethanol, and then centrifuged, repeatedly. The washed sample powders were dried in an oven at 70 °C for two days. For materials A1 (NiOx-500 °C) and B1 (NiOy-500 °C), the dried sample powders were thermally treated at 500 °C in an electric furnace for 4 h. For materials A2 (NiOx@TiO2-500 °C) and B2 (NiOy@TiO2-500 °C), the dried A and B sample powders (0.1 g) were dispersed in ethanol solvent, and 0.5 mL of titanium (IV) isopropoxide  was added to it. While stirring the solution, water vapor was slowly introduced using a humidifier for 2 h. After the reaction, the samples were fully washed with ethanol solvent to remove uncoated TTIP. Then, the collected sample was dried and thermally annealed at 500 °C for 4 h. For materials A3 (NiOx500 °C@TiO2-500 °C) and B3 (NiOy500°C@TiO2-500°C), the A1 and B1 sample powders were used for TiO2 coating. The other procedures were the same as those used for the A2 and B2 samples.Two different Ni oxide complexes—i.e., NiO−1. UV-Vis absorption spectra of the samples were recorded using a Neosys-2000 double beam UV-Vis spectrometer .The morphology of the prepared samples was examined using field-emission SEM . The samples were placed on an HF-etched Si substrate. The crystal phases were identified using powder XRD; the patterns were obtained by using a PANalytical X’Pert Pro MPD diffractometer  equipped with a Cu Kα radiation source. FT-IR spectra were measured using a Nicolet iS 10 FT-IR spectrometer  with an attenuated total reflection mode between 500 and 4000 cm−1 under flow of CO (1%) and O2 (2.5%) mixed gas in N2 balance. The reaction gas products, such as CO2 (mass = 44 amu), were examined using an SRS RGA200 quadrupole mass spectrometer . After the first CO oxidation run, the sample was naturally cooled to room temperature; then, the second CO oxidation run was performed. The electrochemical measurements were conducted using a CHI 660D electrochemical work station  with a conventional three-electrode configuration  in 6.0 M KOH electrolyte solution. For the working electrode, the sample powder (60 wt %) was fully mixed with acetylene black (20 wt %) and poly(vinylidene fluoride) (20 wt %) in 2 mL of N-methyl-2-pyrrolidone solvent. The dispersion was slowly dropped onto Ni foam (1 × 1 cm2), dried, and pressed to fabricate a thin Ni foam sheet. The CVs were obtained between −0.2 and 6.0 V, galvanostatic CD experiments were conducted with potentials ranging from 0.1 to 0.4 V at the charge density of 0.83 A·g−1, and electrochemical impedance measurements were performed over a frequency range from 0.1 MHz to 0.01 Hz.For photocatalytic mixed dye degradation, equal amounts of three different dye  solutions with concentrations of 5 mg/L (=ppm) were mixed to prepare a mixed dye test solution. Then, 20 mg of sample powder was dispersed in 50 mL of the mixed dye solution to achieve adsorption-desorption equilibrium under dark conditions for 1 h. Upon achieving the equilibrium, 2 mL of the solution was taken and centrifuged to remove the residual powder, and a UV-Vis absorption spectrum was recorded by using a V-530 UV-Vis spectrometer . As the equilibrium was reached, the mixed dye solution was examined under UV (or visible) irradiation. For visible light irradiation, a 500 W Halogen lamp (>400 nm) was used and the distance from the lamp and the dye solution in 100 mL beaker (a diameter of 5.5 cm) was fixed at about 30 cm. For UV light irradiation, four 6W UV (365 nm) lamps were used. Every 2 h, 2 mL of the solution was collected to record the UV-Vis absorption spectrum. For the CO oxidation reaction, 10 mg of sample powder was loaded in a flow-type quartz U-tube with an inner diameter of 4 mm. CO oxidation experiments were performed by increasing the temperature up to 500 °C at the rate of 10 °C·min2 core-shell nanostructures. Generally, TiO2 coating on Ni(OH)2 plates and on annealed NiO showed poor photocatalytic, thermocatalytic, and supercapacitor performances. Therefore, it is advisable to use rod-shaped Ni oxalate complex as a starting material for TiO2 coating. The main findings are as follows:2 and Ni oxalate complexes, respectively. NiO crystal phase was commonly obtained by thermal annealing at 500 °C.Nanoplates and nanorods were synthesized by hydrothermal method, and showed XRD patterns of Ni(OH)2 core-shells with interfacial NiTiO3 could be prepared by TiO2 coating on as-synthesized Ni samples, followed by thermal annealing at 500 °C. TiO2 coating on annealed (500 °C) NiO, followed by thermal annealing at 500 °C, showed no XRD detectable NiTiO3 at the interface.NiO@TiO2 coating on Ni oxalate complex followed by thermal annealing (sample B2) showed the best photocatalytic activity for mixed dye degradation under visible light.For photocatalytic dye degradation, TiOy@TiO2-500 °C) was also more efficient for CO oxidation than other samples.For CO oxidation, the B2 sample (NiOx500°C@TiO2-500°C) was more efficient for specific capacitance than other samples. Specific capacitances were obtained to be in the range of 50–320 F/g. When Ni(OH)2 plates were used as starting material, the TiO2 coating showed a specific capacitance of only 50 F/g. However, when rod-shaped Ni oxalate complex was used, the TiO2 coating showed specific capacitance values of 211 and 134 F/g.For the case of specific capacitance, the B3 sample (NiOIn this work, we investigated the preparation method, characteristics, and various application performances of NiO@TiOBriefly, the present study showed that the sample preparation procedures and the annealing steps vary greatly in the application fields to achieve a higher performance. The obtained valuable information on the preparation methods of the investigated core-shell structures could be extended to other transition metal oxide core-shell structures, promoting their development and diverse applications.


The morphology, structure, magnetic hyperthermia ability, drug release behavior, in vitro cytotoxicity and cellular uptake of MMS nanoparticles were investigated. The results revealed that the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles had spherical morphology and average particle sizes of 390 and 320 nm, respectively. The MCN@C/mSiO2 nanoparticles exhibited higher magnetic hyperthermia ability compared to the MCN/mSiO2 nanoparticles, but the MCN/mSiO2 nanoparticles had higher drug loading capacity. Both MCN@C/mSiO2 and MCN/mSiO2 nanoparticles had similar drug release behavior with pH-controlled release and temperature-accelerated release. Furthermore, the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles showed low cytotoxicity and could be internalized into HeLa cells. Therefore, the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles would be promising for the combination of drug delivery and magnetic hyperthermia treatment in cancer therapy.We report the preparation of magnetic mesoporous silica (MMS) nanoparticles with the potential multifunctionality of drug delivery and magnetic hyperthermia. Carbon-encapsulated magnetic colloidal nanoparticles (MCN@C) were used to coat mesoporous silica shells for the formation of the core-shell structured MMS nanoparticles (MCN@C/mSiO  Chemotherapy is often used for cancer therapy, but the side-effect of toxic free anticancer drugs is very serious to the body. ThereforTo date, a variety of carriers including organic and inorganic materials have been studied for anticancer drug delivery. RecentlyHowever, cancer recurrence often occurs when only chemotherapy is used to treat the tumors, although the use of carriers can allow targeted drug delivery and controlled drug release. Therefore, much effort has been made to enhance cancer therapeutic efficacy by combining other efficient approaches, such as gene therapy, immunotherapy, radiation therapy, and magnetic hyperthermia, to achieve a synergistic effect. Among th3O4/FMSMs), which showed a sustained drug release behavior and excellent magnetic responsivity to external magnetic field. Furthermore, the Fe3O4/FMSMs carriers could deliver DOX into the cell cytoplasm and cell nucleus3O4/Fe2O3@mSiO2 nanocapsules with a single or double mesoporous silica shell, and these nanocapsules had sufficient magnetic responsivity and high anticancer drug loading capacity2O3-encapsulated mesoporous silica microspheres, and these magnetic microspheres showed the controlled release of therapeutic drugs and good magnetic heating capacity under a low-frequency alternating magnetic fieldTo solve this critical issue, magnetic mesoporous silica (MMS) nanoparticles are proposed to be a multifunctional platform for drug delivery and magnetic hyperthermia. Mesoporous silica can deliver drugs owing to the high drug loading in mesoporous channels. MNPs can3O4/SiO2 MMS nanoparticles by encapsulating Fe3O4 nanoparticles in mesoporous silica using a sol-gel method, which had pH-controlled DOX release behavior and magnetic heating capacity under an alternating magnetic field.[3O4 amount was limited in the prepared Fe3O4/SiO2 MMS nanoparticles, which results in the lower magnetic heating capacity.Recently, we prepared Feic field. Using DNic field. However,2 nanoparticles. Here, the carbon layer could enhance the ability to protect the magnetic colloidal nanoparticles from oxidation or dissolution in an acidic environment. After the treatment of the MCN@C/mSiO2 nanoparticles by calcination to remove the carbon layers, rattle-type MCN/mSiO2 nanoparticles were obtained. Furthermore, magnetic hyperthermia ability, drug release behavior, biocompatibility and cellular uptake of the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were investigated.In this study, we report a kind of MMS nanoparticle as a multifunctional platform for drug delivery with synergistic magnetic hyperthermia ability. Preparation of MMS nanoparticles involves the synthesis of carbon-encapsulated magnetic colloidal nanoparticles (MCN@C) by solvothermal treatment  and subs2. 2.1. 5H5)2, ≥98%), hydrogen peroxide , acetone , cetyl trimethyl ammonium bromide (CTAB), triethanolamine (TEA) and tetraethyl orthosilicate (TEOS) were of analytic grade from the Sinopharm Chemical Reagent (Shanghai) Co., Ltd, Shanghai, China. Doxorubicin hydrochloride (DOX) was purchased from Sangon Biotech (Shanghai) Co., Ltd . Dodecane was obtained from TCI, Shanghai, China. All chemicals were used as received without further purification.Ferrocene  ethanol solution (0.6 wt%) at 60°C for 6 h thrice to remove the surfactant, which was denoted as MCN@C/mSiO2 nanoparticles. Rattle-type MMS nanoparticles were obtained by the calcination of the MCN@C/mSiO2 nanoparticles at 540°C for 7 h, which was denoted as MCN/mSiO2 nanoparticles.MMS nanoparticles were prepared via a one-pot biphase stratification approach after some modification. Typicall2.4. 2 sorption measurements were carried out on a Micromeritics Tristar 3020 automated surface area and pore size analyzer  at 77 K under continuous adsorption conditions. Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) methods were used to determine the surface area and pore size distribution. Magnetization curves were carried out using a LakeShore 7407 vibrating sample magnetometer  at 298 K.The wide-angle X-ray diffraction (WAXRD) patterns were performed on a D8 ADVANCE powder diffractometer  using Cu Kα1 radiation (1.5405 Å). Scanning electron microscope (SEM) observations were carried out with a FEI Quanta 450 field emission setup. Scanning transmission electron microscopy (STEM) images were obtained with a Tecnai G2 F30 electron microscope  operated at an acceleration voltage of 300 kV. UV-vis analysis was performed on a NanoDrop2000C spectrophotometer . N2.5. 2 and MCN/mSiO2 nanoparticles to verify drug release behavior. For drug loading, DOX was dissolved into PBS to obtain DOX solution with a concentration of 0.5 mg ml–1. Subsequently, 60 mg of MCN@C/mSiO2 or MCN/mSiO2 nanoparticles was dispersed into 12 ml of DOX solution. The mixture solution was agitated for 24 h under dark conditions, and then collected by centrifugation to obtain the DOX-loaded MCN@C/mSiO2 or MCN/mSiO2 nanoparticles. The DOX-loaded nanoparticles were washed with PBS twice to remove DOX adsorbed on the surfaces. The DOX loading amount was determined by measuring the initial and residual DOX solutions using UV-vis analysis at the wavelength of 481 nm.In this study, anticancer drug  was introduced into the MCN@C/mSiO2 or MCN/mSiO2 nanoparticles were performed as follows: 60 mg of the DOX-loaded MCN@C/mSiO2 or MCN/mSiO2 nanoparticles was dispersed in 10 ml of the release medium (pH 5.0 or pH 7.4 at 37°C and pH 5.0 or pH 7.4 at 45°C). At the predetermined time intervals, 20 μl of the released solution was withdrew and centrifuged for UV-vis analysis. After the UV-vis analysis, the residual solution was returned to the release system.The DOX release behaviors from the MCN@C/mSiO2.6. 2 and MCN/mSiO2 nanoparticles was carried out on a DM100 System . The MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were dispersed in H2O, and the concentration was 30 mg ml–1, respectively. Subsequently, 1.0 ml of the nanoparticles suspension was put in the test vessel and then fixed under an alternating magnetic field. The magnetic field strength and frequency were set to be 180 Gauss and 409 kHz, respectively. The specific absorption rate (SAR) was calculated to evaluate the magnetic hyperthermia ability of the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles.The evaluation of magnetic hyperthermia ability of the MCN@C, MCN@C/mSiO2.7. in vitro cytotoxicity and cellular uptake of MMS nanoparticles. HeLa cell lines were maintained in MEM medium containing 10% fetal bovine serum, 100 units ml–1 penicillin, and 100 mg ml–1 streptomycin. Cells were cultured with the complete medium in 5% CO2 at 37°C.In this study, HeLa cells lines were used to test 2.8. In vitro cytotoxicities of the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were evaluated using Cell Counting Kit-8 (CCK-8) assay . Before cell seeding, the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were dispersed in MEM medium with a centration of 1 mg ml–1. HeLa cells were seeded into a 96-well plate at a density of 5000 cells in each well. Subsequently, the nanoparticles suspension were added in each well, and the final concentrations of the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were 0, 25, 50, 75 and 100 μg ml–1 with the medium volume of 100 μl in each well. After 24 h incubation of cells, 10 μl of CCK-8 solution was added into each well, and the cells were incubated for another 3 h. The optical density (OD) was then measured by the absorbance at 450 nm using a microplate reader .2.9. 2 and MCN/mSiO2 nanoparticles, three kinds of nanoparticles were labeled with rhodamine B isothiocyanate (RBITC) to form RBITC-labeled nanoparticles. Typically, 1.0 × 105 cells were seeded in a 35-mm Petri dish. After the cells were attached to the bottom of Petri dish they were washed twice with PBS. Subsequently, RBITC-labeled nanoparticles were added to the Petri dishes (100 μg ml–1). After another 4 h incubation, the cells were washed with PBS three times to remove the remaining nanoparticles and dead cells. Then, the nuclei of cells were stained for 25 min using Hoechst 33342 solution (0.5 μg ml–1). After washing with PBS to remove the residual dye molecules, the cells were fixed with 4% of paraformaldehyde for 20 min. Finally, the cells were washed with PBS for three times, and observed under a confocal laser scanning microscopy .For observation of cellular uptake of the MCN@C, MCN@C/mSiO3. 3.1. 2 and MCN/mSiO2 nanoparticles are shown in Figure 2 nanoparticles, the XRD pattern showed a broad reflection at 2θ = 20–25°, suggesting an amorphous structure of the mesoporous silica coating; but other diffraction peaks were the same as those of MCN@C nanoparticles, indicating that the crystalline structure of the MCN@C nanoparticles did not change after the coating of mesoporous silica and the removal of surfactant. But for MCN/mSiO2 nanoparticles, several diffraction peaks indexed to hematite (JCPDS file 33-664) were observed on the XRD pattern, except for the peaks for magnetite or maghemite, which suggests that some magnetite or maghemite in MCN@C nanoparticles have converted to hematite in MCN/mSiO2 nanoparticles due to the calcination treatment for the removal of carbon and surfactant. On the other hand, compared to the MCN@C and MCN@C/mSiO2 nanoparticles, the width of the diffraction peaks of the MCN/mSiO2 nanoparticles became much narrower, which suggested that the calcination treatment of the MCN@C/mSiO2 nanoparticles induced the formation of larger crystals of magnetite or maghemite. According to Scherrer’s formula, the magnetic nanocrystal size was increased from 16.1 nm in the MCN@C/mSiO2 nanoparticles to 18.2 nm in the MCN/mSiO2 nanoparticles.The wide-angle XRD patterns of MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles measured at 298 K with a vibrating sample magnetometer (VSM). Similar to the previous reports,[Ms) of the MCN@C nanoparticles was estimated to be 34.4 emu g–1. It can also be observed that a very small hysteresis loop is for the MCN@C/mSiO2 nanoparticles and the Ms decreased to be 16.5 emu g–1. This indicated that the MCN@C nanoparticles still maintained superparamagnetic behavior. The decrease in Ms was attributed to the non-magnetic mesoporous silica coating on the MCN@C nanoparticles. However, a relatively obvious hysteresis loop was observed in the magnetization curve of the MCN/mSiO2 nanoparticles. It might be that the larger crystals of magnetite or maghemite and hematite formation contributed the ferromagnetic behavior of the MCN/mSiO2 nanoparticles. On the other hand, the Ms of the MCN/mSiO2 nanoparticles decreased to be 14.7 emu g–1 owing to the formation of some hematite crystals from magnetite or maghemite during the calcination treatment. Compared to our previously reported MMS nanoparticles (Ms of 4.2–8.1 emu g–1),[2 and MCN/mSiO2 nanoparticles exhibited much higher magnetization saturation, which might enhance the magnetic heating capacity.Figure  reports, the magnemu g–1), the MCN@2 and MCN/mSiO2 nanoparticles. The MCN@C nanoparticles had a narrow particle size distribution, and the average particle size was about 170 nm  was about 70 m2 g–1. However, both isotherms of the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles belong to type IV isotherm, indicating the mesoporous structure of the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles. The SBET of the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were 606 and 709 m2 g–1, respectively. The pore volumes (VP) at P/P0 = 0.97 for the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were 0.95 and 1.08 cm3 g–1, respectively  of the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles, which was quantified for the magnetic heating capacity of magnetic materials, was estimated to be 54.7, 34.8 and 25.7 W g–1, respectively. The MCN@C/mSiO2 and MCN/mSiO2 nanoparticles also showed good magnetic hyperthermia ability, although the magnetic hyperthermia ability of the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles was lower than that of the MCN@C nanoparticles, owing to the coating of non-magnetic mesoporous silica on the MCN@C nanoparticles. On the other hand, the MCN@C/mSiO2 nanoparticles showed a little higher magnetic heating capacity than the MCN/mSiO2 nanoparticles, which is attributed to the maintaining of iron oxide crystals in the MCN@C/mSiO2 nanoparticles during extracting treatment, but a small amount of hematite crystals were formed in the MCN/mSiO2 nanoparticles during the calcination treatment.Figure 3.3. 2 and MCN/mSiO2 nanoparticles in this study. The DOX loading amounts in the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were 58.5 and 96.2 μg DOX per 1 mg of the MCN@C/mSiO2 or MCN/mSiO2 nanoparticles, respectively. Here, the higher DOX loading capacity of the MCN/mSiO2 nanoparticles was attributed to its higher surface area and hollow cavity.Doxorubicin hydrochloride (DOX) was used as a model anticancer drug to investigate the drug release behavior from the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles in the release media with pH 7.4 and 5.0 at temperatures of 37 and 45°C. Here, the temperature of 45°C was used to evaluate the possibility of drug release in the range of hyperthermia temperature. Both MCN@C/mSiO2 and MCN/mSiO2 nanoparticles exhibited pH dependent DOX release behavior. The DOX releases from the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were very slow in pH 7.4 medium (only c.2–5% of DOX release in 8 h), but the DOX release became faster in pH 5.0 medium (c.25–30% of DOX release in 8 h). The DOX release behaviors from the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles are similar to those in previous reports on the DOX release from mesoporous silica nanoparticles.[Figure articles. It has b2 and MCN/mSiO2 nanoparticles, the DOX release at 45°C was slightly faster compared to that at 37°C, which might be because the increased temperature can accelerate the diffusion of DOX molecules in the mesoporous channels. In this study, the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles can generate heat under an alternating magnetic field to raise temperature, and thereby stimulate drug diffusion.On the other hand, for both MCN@C/mSiOTherefore, such drug release behavior could decrease the side-effect of toxic anticancer drug delivery, because anticancer drugs mostly do not release from the carriers during the circulation of the drug delivery system in the blood stream (pH 7.4). When the drug delivery system is internalized in the targeted cancer cells, faster DOX release could enhance the therapeutic efficiency owing to the acidic environment in the endosome/lysosome and cytosol of cancer cells (pH 5.0–5.5). The combination with magnetic hyperthermia could further accelerate drug release after the endocytosis of drug delivery system by cancer cells.3.4. in vitro cytotoxicities of the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles against HeLa cells evaluated using CCK-8 assay. Cell viabilities of the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles were more than 90% even up to a concentration of 100 μg ml–1, and they had no significant difference compared to those of the control group. The results revealed that the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles are safe for drug delivery.Figure 2 and MCN/mSiO2 nanoparticles were incubated with HeLa cells for 4 h to observe the cellular uptake. As shown in Figure 2 and MCN/mSiO2 nanoparticles were taken up into HeLa cells after endocytosis. However, the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles have not been internalized into the nuclei. Furthermore, the distributions of red fluorescence had no significant difference among the MCN@C, MCN@C/mSiO2 and MCN/mSiO2 nanoparticles. Therefore, the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles as nanocarriers for anticancer drug delivery could achieve local drug delivery and magnetic hyperthermia, and thereby enhance the cancer therapeutic efficiency.Cellular uptake of the drug carriers is very important for enhancing the drug delivery efficiency. On the one hand, cellular uptake of drug carriers could be beneficial for magnetic hyperthermia therapy owing to magnetic local heating in cancer cells. On the other hand, cellular uptake of drug carriers could also enhance the chemotherapeutic efficiency due to the intracellular delivery of drugs. In this study, RBITC-labeled MCN@C, MCN@C/mSiO4. 2 and MCN/mSiO2) as a multifunctional platform for potential drug delivery and magnetic hyperthermia. The MCN@C/mSiO2 nanoparticles had core-shell structure with higher magnetic heating capacity compared to the MCN/mSiO2 nanoparticles, but the MCN/mSiO2 nanoparticles had higher drug loading capacity. Both MCN@C/mSiO2 and MCN/mSiO2 nanoparticles had similar drug release behavior with pH-controlled release and temperature-accelerated release. Furthermore, the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles showed low cytotoxicity and could be internalized into cells within a short period. Therefore, the MCN@C/mSiO2 and MCN/mSiO2 nanoparticles would be promising for chemotherapy with synergistic magnetic hyperthermia in cancer therapy.In this study, we have synthesized MMS nanoparticles (MCN@C/mSiONo potential conflict of interest was reported by the authors.


Amino acid availability stimulates protein synthesis via the mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway. In response to an increase in cellular amino acid availability, translocation of cytosolic mTORC1 to the lysosomal surface is required to stimulate mTORC1 kinase activity. However, research elucidating the amino acid responsive mechanisms have thus far only been conducted in in vitro models.Our primary objective was to determine whether an increase in amino acid availability within human skeletal muscle in vivo would alter the expression of genes associated with amino acid sensing, transport and mTORC1 regulation. Our secondary objective was to determine whether an acute perturbation in lysosomal function would disrupt the normal pattern of muscle amino acid responsive gene expression.vastus lateralis taken before and after EAA ingestion. We determined the relative mRNA expression of 51 potential amino acid responsive genes using RT-qPCR.We recruited 13 young adults into one of two groups: The first group ingested 10 g of essential amino acids (EAA). The second group ingested 10 g of EAA in the presence of chloroquine (CQ), a lysosomotropic agent. The subjects from each group had biopsies of the P < 0.05) in response to EAA ingestion . In the CQ group, EAA ingestion resulted in a differential expression as compared to EAA alone  between the two groups.)There was a differential mRNA expression for 22 genes, with 15 mRNAs significantly changing  contains supplementary material, which is available to authorized users. The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis. mTORC1 exists as a dimer and consists of the core subunits mTOR, Raptor (regulatory-associated protein of mTOR), mLST8 , and noncore regulatory subunits DEPTOR (DEP Domain Containing MTOR-Interacting Protein) and PRAS40 (Proline-rich AKT1 substrate 1) –5. DurinAmino acid availability is a potent anabolic stimulator of mTORC1 activity . How celMuch of the work characterizing amino acid sensing has used cultured HEK-293 T kidney cells as the model. In vitro work often does not translate to in vivo conditions, particularly in complex tissues such as skeletal muscle. Therefore, the primary aim of the current study was to determine whether genes known to regulate amino acid sensing in vitro would be responsive to an increase in amino acid availability in vivo. Previous work in muscle cells has shown a responsiveness of some amino acid transporter genes to amino acid feeding . We hypoStudies were conducted under protocols approved by the University of Texas Medical Branch Institutional Review Board, written informed consent was obtained from all subjects, in compliance with the Helsinki Declaration of 1975 as revised in 1983. All subjects self-reported during screening as having normal recreational levels of activity. Subjects reporting an exercise training regimen of more than 2× per week were excluded. DEXA  assessed body composition. Exclusion criteria included: diabetes, any active diet or large weight loss, diseases of lung, liver, neurologic, blood, cardiovascular or kidney, history of cancer, current infectious disease, dementia or other significant psychiatric disease, and obesity. This work was a part of a larger study registered as NCT00891696 at clinicaltrials.gov; only information pertinent to the current study is imparted herein.n = 6, 5 males and 1 female) or 10 g ingestion of EAA in the presence of chloroquine . There was no difference (P > 0.05) in the age , BMI  or body fat percentage  between the two groups.We recruited, screened and consented young adults into one of two groups: 10 g ingestion of essential amino acids  period. Following the muscle biopsy, each subject ingested the same 10 g of EAA. One hour after EAA ingestion, a second muscle biopsy was obtained (post-EAA), as this time point coincides with peak EAA availability and maximal mTORC1 signaling.The evening prior to the study the participants received a standardized meal based upon their body mass and were then fasted overnight. Subjects in the CQ group received an oral dose of 250 mg of CQ the night before the study and then a second dose of 500 mg first thing in the morning on the study day (time = 0 h). The prescribed dosage of chloroquine  was the maximum allowed as defined by the study physician, was similar to the therapeutic dosage used to treat malaria, and was consistent with a previous study on protein metabolism , 21, 22.We obtained vastus lateralis muscle biopsies pre- and post-intervention using aseptic techniques, as previously described . BrieflyWe performed RNA isolation and cDNA production similarly to what has been previously reported by our lab . Brieflywww.bio-rad.com). Each plate contained 10 target genes and 2 reference genes . The target genes  were: SLC38A1, SLC38A2, SLC38A4, SLC38A5, SLC38A6, TFEB, SLC38A7, SLC38A8, SLC38A9, SLC38A10, DDIT4, DDIT4L, LAMP2, MAP1LC3A, RHEB, SLC36A1, SLC36A2, SLC7A1, SLC7A5, SLC7A8, ATP6VOD1, C7orf59, HBXIP, LAMTOR1, LAMTOR2, LAMTOR3, RRAGA, RRAGB, RRAGC, RRAGD, DEPDC5, FLCN, FNIP1, MIOS, NPRL2, NPRL3, SEC13, SEH1L, WDR24, WDR59, AKT1S1, DEPTOR, LARS, MLST8, mTOR, PIK3C3, RPTOR, TBC1D7, TSC1, and TSC2. There was no difference in expression of the two reference genes (B2M and GAPDH) from the basal to fed condition (P > 0.20).We designed five different custom 96-well plates that were prepared and validated/optimized in house by BioRad  and was considered stable.The SESN2 (SESTRIN2) and the two reference genes, B2M and GAPDH, primers were self-designed and purchased from SIGMA. See Table n = 6 for CQ and 5 for EAA, as adequate tissue was not obtained from 2 of the enrolled female subjects in the EAA group). For the PrimePCR plates: 10 μl of SYBR green supermix , 3 μl of template (from 1:8 dilution of cDNA with DNase/RNase free water), and 9 μl RNase-free water were loaded into wells, in duplicate for each subject sample, on the PrimePCR plate. The plate was sealed, spun down on a centrifuge, and then loaded into the CFX cycler. The following protocol was used: 1 cycle for 2 min @ 95 °C; then 40 cycles of: 95 °C for 5 s, 60 °C for 30 s; followed by 95 °C for 10 s and a melt curve. Results were quantified using CFX connect software (BioRad) and then the 2-∆∆Ct method was used on the Cq (quantification cycle) to calculate the final mRNA fold-change values from pre-EAA to post-EAA using the geometric mean of the two reference genes and reported according to MIQE guidelines [We determined the relative mRNA expression of the 51 potentially amino acid responsive genes using RT-qPCR (BioRad CFX) on pre-EAA and post-EAA muscle biopsy samples from 11 of the 13 study participants  and 2x2 Repeated Measures ANOVA  used p < 0.05 for significance, with trends reported at p < 0.10. Statistical software package was SPSS v.23 (IBM).Data is presented as means ± SE, or as % change ± SE, as appropriate. Student’s paired P < 0.05). In EAA, 6 more also had increases: SLC7A8, NPRL3, TSC2, SLC36A1, LAMTOR2, and MAP1LC3; but had p-values P < 0.10 and >0.05--heretofore referred to as genes of interest. In the group that ingested EAA and chloroquine (CQ), 6 mRNAs changed expression: SLC38A10, SLC7A1, TBC1D7, and TSC2 all decreased (P < 0.05); while DDIT4L and NPRL3 both increased (P < 0.05). CQ had 4 genes of interest: SLC38A9, DEPDC5 and LAMP2 increased while LAMTOR3 decreased (P < 0.10 and >0.05). There were significant differences (P < 0.05) between the fold changes in the two treatment groups in 11 different genes . In addition, between the groups there were trend (P < 0.10 and >0.05) differences in 7 other genes: SLC7A1, TBC1D7, TSC1, SLC38A4, LAMTOR1, LAMTOR2, and FLCN all showed a greater fold change in EAA. The genes differentially expressed and the genes of interest coded for a variety of different mRNAs that translate into proteins/protein complexes that are both positive and negative regulators of mTORC1 activity (20), amino acid transporters (6), autophagy genes (2), mTORC1 subunits (4), and lysosomal formation/maintenance proteins (2) (some mRNAs belong to more than one category). Additional file p-values. Additional file n = 3 in CQ) are delineated by darker data points.In order to determine the effect of EAA ingestion on gene expression, we measured the expression levels of 51 genes involved in processes including amino acid sensing, amino acid transport and mTORC1 regulation. There were significant changes in 22 of the 50 genes tested from pre- to post-EAA. In the normal feeding scenario (EAA), expression of 15 mRNAs changed: SLC38A10, SLC38A9, C7orf59, WDR24, MIOS, DDIT4, DPEDC5, NPRL2, TSC1, AKT1S1, MLST8, mTOR, RPTOR, PIK3C3 and TFEB all increased (significantly affected by the treatments (P < 0.05). SLC38A10 increased 57 ± 15% in EAA, but decreased −20 ± 9% in CQ; SLC38A9 increased 31 ± 9% in EAA; SLC7A1 decreased −37 ± 11% in CQ; and both SLC7A8 and SLC38A10 had higher expression in EAA than CQ : DEPDC5 20 ± 3% and NPRL2 12 ± 2% in EAA, and NPRL3 15 ± 5% in CQ. All three of the mRNAs coding genes from the GATOR2 complex (positive regulator of mTOC1) showed a significant change (P < 0.05): MIOS 16 ± 4% and WDR24 21 ± 4% increasing in EAA, and WDR59 higher in EAA than CQ  between EAA and CQ, with DDIT4 (REDD1) mRNA expression increasing 209 ± 35% in EAA and DDIT4L (REDD2) mRNA increasing by 18 ± 5% in CQ  subunits’ mRNA expression was differentially expressed by treatment, with TSC1 increasing 15 ± 2% in EAA, and TSC2 (−19 ± 7%) and TBC1D7 (−8 ± 3%) decreasing in CQ Fig. . Genes c CQ Fig. . In addiP < 0.05) after EAA ingestion in the EAA group, but were not changed (P > 0.05) in the CQ group  increased (P = 0.32), but there was a trend for a 63.7 ± 29.0% increase in SESN2 mRNA expression in the EAA group, P = 0.09. There was quite a bit of variance in the EAA group with 3 subjects having ~2-fold increases in expression and 2 non-responders. The EAA group had a trend for a higher mRNA expression of SESN2 in response to EAA in comparison with the CQ group (P = 0.071). See Additional file There was no change is SESN2 mRNA expression from pre to post EAA ingestion in the CQ group  in EAA, but the difference between EAA and CQ was significant . LAT2 is a neutral amino acid antiporter that is similar to LAT1 in that it pumps out nonessential amino acids (glutamine) and pumps in neutral essential amino acids (leucine), acting as a critical regulator of leucine concentration in the cell. The only difference in LAT1 and LAT2 is in the composition of the light subunit, as both also contain the heavy subunit CD98, with the LAT2 subunit (SLC7A8) having a broader substrate range [We examined the expression levels of numerous amino acid transporters due to their close relationship with protein synthesis and mTORC1 signaling. In response to an increase in amino acid availability both SLC38A10 and SLC38A9 mRNA expression increased in the EAA group, with both SLC38A10 and SLC7A1  decreasing in the CQ group. Both SLC38A9 and SLC38A10 are sodium-coupled neutral amino acid transporters, with SLC38A9 serving the function as lysosomal lumen arginine sensor contributing to activation of RAGULATOR. SLC38A10 has not been fully characterized, but is believed to have a similar function . SLC7A8 te range .P = 0.063, due to the presence of 1 negative outlier: without the outlier it increased 37 ± 5%, P = 0.005). Therefore, while there were some differences in mRNA expression of particular transporters from prior studies, the important thing to note is that a number of transporters were affected by an increase in amino acid availability, suggesting that the amino acid transport system is sensitive not only to changes in amino acid concentrations, but also to the functional status of the lysosome.Previous work discovered gene expression of some amino acid transporters significantly increased at 1-h post-EA . These tPrevious work has established the role of both amino acid sensing and nutritional regulation in mTORC1 activation , 5, 8. OTSC complex genes increased in EAA (TSC1), but decreased in CQ (TSC2 and TBC1D7). In addition, AKT1A1 (PRAS40) and DDIT4 (REDD1) mRNA expression also increased in the EAA group. Low oxygen levels in the cell are detected via Redd1  or Redd2 , activating the TSC complex . There is evidence that REDD1 and REDD2 act to modulate mTORC1 signaling by adjusting gain to limit output and that the proteins are responsive to both nutrient levels and exercise . The TSCMore negative regulators (10) of mTORC1 activity increased with EAA than positive regulators (5). The upregulation of negative regulator genes may indicate the existence of a negative feedback loop.There are a few limitations with our study that should be discussed. First, the total amount of chloroquine that was administered was relatively low. This was done in order to ensure participant safety. However, this may have resulted in an incomplete inhibition of lysosomal function. Also, since we did not independently measure lysosomal activity, we can only assume that lysosomal function was perturbed at least partially by chloroquine. Second, we did not measure protein expression as it is unlikely that we would be able to detect a change in protein expression within the one-hour time frame of our intervention. In addition, much of the transient changes in mTORC1 activation is driven by covalent modification of proteins rather than by overall protein content. The changes in mTORC1 signaling post-amino acid ingestion has been previously well documented , 15, 27,In our in vivo study, we provide preliminary evidence to support our hypothesis that a discernable pattern of gene expression occurred in response to an acute increase in amino acid availability. In addition, the normal gene expression response to amino acid availability is partially disrupted by the administration of chloroquine. In fact, in 19 of the 22 genes found to have been significantly altered, the administration of CQ resulted in a differential mRNA expression pattern . This finding highlights the importance of the lysosome in amino acid response in the in vivo setting. We acknowledge that mRNA expression at a single time point is only a snapshot of a dynamic system. Based upon these findings, future work may confirm the link between amino acid response and mTORC1 translocation to the lysosome. Thus, future mechanistic work in muscle cells and in transgenic mice are required to determine definitively whether the amino acid sensing mechanisms identified in vitro are functional in skeletal muscle.


Reactive astrogliosis is one of the significantly pathological features in ischemic stroke accompanied with changes in gene expression, morphology, and proliferation. KCa3.1 was involved in TGF-β-induced astrogliosis in vitro and also contributed to astrogliosis-mediated neuroinflammation in neurodegeneration disease.−/− mice were subjected to permanent middle cerebral artery occlusion (pMCAO) to evaluate the infarct areas by 2,3,5-triphenyltetrazolium hydrochloride staining and neurological deficit. KCa3.1 channels expression and cell localization in the brain of pMCAO mice model were measured by immunoblotting and immunostaining. Glia activation and neuron loss was measured by immunostaining. DiBAC4 (3) and Fluo-4AM were used to measure membrane potential and cytosolic Ca2+ level in oxygen-glucose deprivation induced reactive astrocytes in vitro.Wild type mice and KCa3.1−/− mice exhibited significantly smaller infarct areas on pMCAO and improved neurological deficit. Both activated gliosis and neuronal loss were attenuated in KCa3.1−/− pMCAO mice. In the primary cultured astrocytes, the expressions of KCa3.1 and TRPV4 were increased associated with upregulation of astrogliosis marker GFAP induced by oxygen-glucose deprivation. The activation of KCa3.1 hyperpolarized membrane potential and, by promoting the driving force for calcium, induced calcium entry through TRPV4, a cation channel of the transient receptor potential family. Double-labeled staining showed that KCa3.1 and TRPV4 channels co-localized in astrocytes. Blockade of KCa3.1 or TRPV4 inhibited the phenotype switch of reactive astrogliosis.Immunohistochemistry on pMCAO mice infarcts showed strong upregulation of KCa3.1 immunoreactivity in reactive astrogliosis. KCa3.1Our data suggested that KCa3.1 inhibition might represent a promising therapeutic strategy for ischemia stroke.The online version of this article (10.1186/s12974-017-0973-8) contains supplementary material, which is available to authorized users. Briefly2+ ([Ca2+]i) was measured as described previously [2+]i) by monitoring Fluo-4 fluorescence. TRAM-34 (1 and 10 μM) or HC 067047 (10 μM) was added 1 h before the experiment. Fluo-4 fluorescence was measured at 510 nm, with excitation at 488 nm. Confocal images were taken and stored every 1 s for 360 s.Cytosolic Caeviously . Brieflyt test when comparing between two groups, or the non-parametric Mann–Whitney test was applied. Statistical significance was set at p < 0.05.All data are presented as means ± SEM. Statistical analyses were performed using Prism software . Data were tested for Gaussian distribution with the Kolmogorov–Smirnov normality test and then analyzed by one-way ANOVA and Dunnett’s post hoc tests. Data were analyzed with unpaired, two-tailed Student’s Most recently, we reported KCa3.1 was increased in reactive astrocytes in the brains of both mouse models of AD and AD patients, and pharmacological inhibition or genetic deletion of KCa3.1 reduced astrogliosis-induced neuronal loss in the AD mouse model . Based o+ neurons in the brains of both control and 6 h pMCAO mice  mice to 3, 6, and 24 h of pMCAO and assessed infarct volumes and neurological deficits  vs 37.5% (6/16), Fig. In order to study the role of KCa3.1 deficiency on the pathological processes of ischemic stroke, we subjected wild type (WT) and KCa3.1 gene deletion  was used to activate KCa3.1 i increase induced by 200 μM 1-EBIO was attenuated in both the 1 μM  or HC 067047 (10 μM), upregulation of GFAP was attenuated , and TRPV4/BK functional coupling regulated bladder contractility in the storage phase i levels to drive phenotype switch of astrogliosis during ischemia stroke. Gene deletion of KCa3.1 attenuated infarct area and reduced neuron loss and gliosis in pMCAO, which may have therapeutic utility in ischemia stroke associated with abnormally high [Ca2+]i in astrogliosis.In conclusion, these data suggested that KCa3.1 and TRPV4 channels forms a signaling complex involved in maintaining abnormally high [CaAdditional file 1:Figure S1. Cerebral blood flow (CBF) of ischemic brain hemisphere before and during permanent middle cerebral artery occlusion (pMCAO) was monitored by transcranial laser Doppler. The arrow depicted the start of pMCAO. (TIFF 58 kb)Additional file 2:Figure S2. Western blot analysis of lysates from 10-week-old male WT mice following 1, 3, 6, or 12 h of pMCAO analyzed by antibodies to KCa3.1 (A) and GFAP (B). (TIFF 223 kb)Additional file 3:Figure S3. GFAP+ reactive astrocytes and Iba1+ activated microglia from the cortex and hippocampal regions of WT or KCa3.1−/− mice brain at 6 h after pMCAO were visualized by immunostaining. Scale bar: 200 μm. WT, wild type. (TIFF 7211 kb)