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Autophagy receptor p62/SQSTM1 promotes the assembly and removal of ubiquitylated proteins by forming p62 bodies and mediating their encapsulation in autophagosomes. Here we show that under nutrient-deficient conditions, cellular p62 specifically undergoes acetylation, which is required for the formation and subsequent autophagic clearance of p62 bodies. We identify K420 and K435 in the UBA domain as the main acetylation sites, and TIP60 and HDAC6 as the acetyltransferase and deacetylase. Mechanically, acetylation at both K420 and K435 sites enhances p62 binding to ubiquitin by disrupting UBA dimerization, while K435 acetylation also directly increases the UBA-ubiquitin affinity. Furthermore, we show that acetylation of p62 facilitates polyubiquitin chain-induced p62 phase separation. Our results suggest an essential role of p62 acetylation in the selective degradation of ubiquitylated proteins in cells under nutrient stress, by specifically regulating the assembly of p62 bodies., The autophagy receptor p62 mediates the assembly and removal of ubiquitylated protein aggregates by forming p62 bodies. Here, the authors identify an acetylation-dependent mechanism that regulates formation and autophagic clearance of p62 bodies under nutrient-deficient conditions.

Objectives α2-adrenergic receptors are reportedly involved in cancer cell proliferation, invasion, and apoptosis through regulation of diverse molecules, which implies that it contributes to tumor progression. However, the functional significance of α2-adrenergic receptors in osteosarcoma (OS) is unclear. Tizanidine hydrochloride (THC), an α2-adrenergic receptor agonist, is often used to alleviate symptoms of spasticity. This study investigated the functional implications of THC treatment on human OS cells and the underlying mechanisms of resulting changes. Methods The proliferation of U2 OS cells was assessed by Cell Counting Kit-8; the migration and invasion capacities of U2 OS cells were then analyzed by transwell assay. Moreover, apoptosis in U2 OS cells was evaluated by flow cytometry and western blot analyses. Additionally, expression levels of key proteins in the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were measured. Results THC inhibited the proliferation, migration, and invasion of U2 OS cells, but promoted apoptosis within these cells. Expression levels of p-AKT, p-mTOR, and p-P70S6K were reduced by exposure to THC, suggesting involvement of PI3K/AKT signaling in THC-induced cytotoxicity within OS cells. Conclusions THC may play a novel role in OS cell cytotoxicity, and these findings suggest a potent therapeutic strategy for OS treatment.

Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. However, for proteins with native cysteines, site-specific attachment may only utilize functional groups orthogonal to sulfhydryl chemistry. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. Demonstrated with ubiquitin and cysteine-containing enzyme EIIB, we show that large PCSs of distinct profiles can be generated for each tag/lanthanoid combination. The DTTA-based lanthanoid tags are associated with large magnetic susceptibility tensors owing to the rigidity of the tags. In particular, introduction of the DTTA-C3 tag affords intermolecular PCSs and enables structural characterization of a transient protein complex between ubiquitin and a UBA domain. Together, we have expanded the repertoire of paramagnetic tags and the applicability of paramagnetic NMR.

To investigate the joint health status of patients with hemophilia treated on-demand and to analyze the incidence trend of hemophilic arthritis, so as to probide the scientific clinical data for furture study.The clinical data of patients with hemophilia admitlted in Tianjin municipal first central hospital form March 2016 to October 2017 were collected, the basic information of patients was recorded; the joint health status was evaluated by using the hemophillia Joint Health Score (HJHS) 2.1; the life guality of patients was analyzed by using the MOS item short form-36 health survey, SF-36; the correlation of joint function with life guatity was analyzed by pearson correlation test.196 ont of 210 patients with hemophila were treated on demand. The average age of patients was 27.81(2-73) years old, Among 196 patients, 189 was hemophilia A (96.43%) and 9 was hemophilia B (3.57). The patients without joint involvement, patients with 1 joint and 32 jionts involvement accounted for 3.57%, 11.72% and 84.71% respectively. The incidonce of involvement in elbow, knae and ankle joints was 71.93%, 80.61% and 82.91% respectively. The joint invlvement rate in patients with mild, intermediate and severe hemphilia accounted for 61.66%, 72.40% and 80.73% respectively. The mean HJHS in intermediate and severe henophilia patients was 23.59±17.02 scores and 26.69±17.68 scores respectively, there was no statistical difference (P0.05). The joint fanction in hemophilia patients negatively correlated with life gnality of patients (r = 0.076).The incidence of arthritis in hemophilia patients is high, and at least 1 joint has been involved in patients aged over 10 years old, moreover the multiple joint involvement exrsts in most patients. The impairment of joint function affects the routine behavious and activities.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD). In the brain, NAMPT is primarily expressed in neurons and can prevent neuronal degeneration. NAMPT is also highly expressed in inflammatory cells, and is responsible for their activation. Since inflammation following traumatic brain injury enhances neuronal damage, we assessed the effects of nicotinamide mononucleotide (NMN), the direct NAMPT metabolite, and FK866, a potent NAMPT inhibitor, on brain injury in a cryoinjury mouse model. Twenty-four hours after brain cryoinjury, the density of neuron and the level of NAD decreased. Both NMN and FK866 alleviated the neuronal loss and decreased the lesion volume. NMN prevented the cryoinjury-induced decrease of NAD level, and FK866 decreased it further. On day 14 after cryoinjury, further neuronal loss occurred, astrocytes and Iba1-positive macrophage/microglia activated, and the NAD level increased. At this time-point, NAMPT expression was strongly induced in Iba1-positive macrophages/microglia in the lesion core. NMN and FK866 also alleviated the neuronal loss and decreased the lesion volume. In addition, FK866 significantly attenuated the activation of astrocytes and Iba1-positive macrophages/microglia, and decreased the NAD, while NMN had no such effects. Taken together, both FK866 and NMN attenuate traumatic brain injury. However, FK866 acts via the inhibition of the NAMPT activity in inflammatory cells resulting in the inhibition of inflammation, whereas NMN is effective via replenishing NAD.

In this paper, superconducting electromagnetic suspension (EMS) technology is studied for the improvement of manufacturing accuracy in NC machine tools. Superconducting EMS feed drive schemes are proposed to separate the moving parts of machine tools from conventional mechanical guide rails. The realization of superconducting EMS technology in machine tools was studied elaborately. A superconducting EMS scheme is proposed, combining the superconducting electromagnets with the double side normal-conducting electromagnets, to achieve high accuracy suspension as well as low power consumption of electromagnets, and a coordination working pattern of the hybrid magnets is designed for the proposed scheme working under varying conditions. Detailed working principle of the coordination pattern is presented. A prototype of superconducting EMS feed drive system was constructed based on the saddle moving part of a gantry machine tool to verify the feasibility and effectiveness of the proposed scheme. The prototype realized the vertical suspension accuracy around 5 μm, and the electromagnet power consumption was greatly reduced under varying working conditions.

To compare emu necrotic femoral head micro structure repaired in two different methods.Fifteen adult emus were divided into 3 groups (all n=5), and the right femoral head was selected to research. The first group was the control group; in the second group, femoral head necrosis was made by cryogen with liquid nitrogen; and in the third group, femoral head necrosis was made by local pure ethanol injection. Right femurs were taken for micro CT examination,then femoral head micro structures were compared among these three groups.No infection or unexpected death was found in all groups. Compared with normal group, necrotic femoral heads in cryogen group showed that bone mineral density significantly reduced after repaire (P=0.015), trabecular space significantly reduced (P=0.001), bone volume fraction significantly enlarged (P=0.036), bone surface/volume fraction (P=0.032) and trabecular numbers (P=0.002) significantly enlarged; trabecular thickness showed no significant difference (P=0.060). Compared with control group, necrotic femoral heads in ethanol group showed that bone mineral density significantly enlarged after repaire (P=0.001), trabecular thickness (P=0.003) and bone surface/volume fraction (P=0.022) significantly enlarged, trabecular space (P=0.001) and bone volume fraction (P=0.001) significantly reduced; the trabecular numbers showed no significant difference (P=0.143). Compared with ethanol group, necrotic femoral heads in cryogen group showed significant lower bone mineral density after repair (P=0.001), significantly lower bone volume fraction (P=0.001), significantly lower trabecular thickness (P=0.001), significantly higher bone surface/volume fraction (P=0.022) and higher trabecular numbers (P=0.003); the trabecular space showed no significant difference (P=0.398).Different repair methods make reconstructed femoral head weight bearing area have different bone structure and bone mineral density, along with different bone trabecular quality.

Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine β-strands (β-stands 1-9) that carries the mature fluorophore and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications.

Functional ceramics naturally have inferior mechanical properties. This study shows that incorporating Ag into lead zirconate titanate (PZT) piezoelectric ceramics by co-firing in air at 1200 °C is effective for mechanical property enhancement. The Ag addition has increased the fracture strength σf from 69 MPa for monolithic PZT to 129 MPa for PZT–15% Ag composite, and increased the corresponding fracture toughness KIC from 1.0 to 2.4 MPa m1/2. The Vickers hardness Hv decreased with increasing Ag concentration, from 3.8 GPa for monolithic PZT to 2.7 GPa for PZT–15% Ag composite. The strength enhancement is attributed to the combined effects of good interface bonding between Ag and PZT, ductile deformation of Ag particles and particle-dispersion strengthening. The fracture toughness enhancement is correlated to crack deflection and crack arrest due to the existence of Ag particle. The enhanced mechanical properties make the PZT/Ag composites promising candidate for piezoelectric actuator applications.

Based on the viscosity of individual crude and its fraction in blends, the viscosity of crude blend was calculated with a mathematic model. Ten previous published empirical models including Arrhenius-type model, Bingham model, Cragoe model and so on, were reviewed and discussed. The study used 654 sets of viscosity data of 70 crude blends to verify these ten published models, with 14 crude oil samples from Xinjiang were involved. Among the ten models, the modified Cragoe model agreed best with the experimental data, which needs the input of the viscosity of every binary blend at a mixing ratio of 1:1. Its absolute average relative deviation is 7.7%. Furthermore, based on these data and the reported models, three new empirical models were developed. The new ones are available without the viscosity of every binary blend at a mixing ratio of 1:1. Their predicting data agree well with the experimental data when the absolute average relative deviations from 12% to 17%.

In order to achieve high performance as well as low power loss in high demanding fields, a space vector modulation based hysteresis current controller is optimized in this paper. The paper analyzes the effects of the voltage space vectors on the current error variations, and proposes the principles to optimize the switching pattern. An optimized switching diagram is constructed. Simulation studies are conducted for a permanent magnet synchronous motor in MATLAB/Simulink. The simulation results demonstrate that the proposed method achieves fast dynamic response, improves the tracking performance and reduces the switching frequency significantly under different conditions.

A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition. DOI: http://dx.doi.org/10.7554/eLife.05767.001, eLife digest Proteins can be tagged with other molecules that indicate what the cell should do with that protein. For example, proteins tagged with a small protein called ubiquitin—which is linked to other ubiquitin molecules to form ‘polyubiquitin’—may be destroyed or relocated within a cell. Like all proteins, a ubiquitin is made up of chains of amino acids. Specific amino acids form the linkages between individual ubiquitins to form a polyubiquitin, and the nature of these linkages influences the effect that a polyubiquitin has on the tagged protein. One linkage involves a lysine amino acid at position 63 (known as Lys63). This linkage is found in the polyubiquitin that is involved in repairing damaged proteins and relocating target proteins to a part of the cell where they are utilized for immune response. To perform these different roles, the polyubiquitin must be able to distinguish between a variety of target proteins. The shape that a protein takes on determines how it works, and most proteins constantly and rapidly switch between different shapes. Previous work suggested that the Lys63-linked polyubiquitin could only take on an elongated ‘open’ structure by itself. It was not clear whether the protein could take on a compact ‘closed’ structure without first binding to a target protein. Liu et al. used a technique known as nuclear magnetic resonance (NMR) to explore the high-resolution structures of the Lys63-linked ubiquitin chain when they are not bound to other proteins. The results showed that a large percentage of the protein was in a closed state, and that there were at least one open shape and two kinds of closed shapes. Liu et al. suggest that the shape of the unbound Lys63-linked ubiquitin chain determines what other proteins can be bound, and that the binding stabilizes the shape of the ubiquitin. This mechanism of binding is known as conformational selection. Further work is required to analyze whether other polyubiquitin chains recognize their partners in a similar manner. DOI: http://dx.doi.org/10.7554/eLife.05767.002

B cell translocation gene 2 (BTG2) has been reported to be a potential tumor suppressor in many types of tumors. However, the roles and molecular mechanisms of BTG2 in osteosarcoma progression are still unknown. In this study, we investigated the role of BTG2 in proliferation and metastasis of osteosarcoma and the underlying mechanism. BTG2 expression levels were measured in fresh osteosarcoma tissues and cell lines. The effects of BTG2 on cell proliferation, migration and invasion were explored by MTT, transwell assays, western blot, and in vivo tumorigenesis in nude mice. We found that BTG2 was down-regulated in human osteosarcoma tissues and cell lines. Overexpression of BTG2 inhibited the proliferation and migration/invasion of human osteosarcoma cells in vitro, it also markedly inhibited xenograft tumor growth in vivo. Furthermore, BTG2 significantly decreased the expression of phosphorylated PI3K and AKT in osteosarcoma cells. Taken together, our data indicate that BTG2 might suppress the tumor growth and metastasis via PI3K/AKT signaling pathway, implying that BTG2 may serve as a potential molecular target for the treatment of osteosarcoma.

To establish a new animal model of osteonecrosis of the femoral head by local ethanol injection in emu.Eight milliliter ethanol was injected slowly to the operated femoral head with customized probe in twenty adult male emus. Postoperatively, hip magnetic resonance imaging was performed at 1, 4, 8, 12 weeks. After emus were sacrificed, the femurs were collected for micro-computed tomography and histological analysis.No emu demonstrated signs of infection or died unexpectedly. Magnetic resonance imaging examination showed broad edema at proximal femur at 1(th) week, and the edema decreased with time, till local edema at femoral head at the 12(th) week. Histological images showed human-like osteonecrotic changes with active bone repair. There were significant differences in trabecular structure and bone mineral density between the operated and intact femoral heads. No collapse was found 6 months after the operation.This emu model of femoral head osteonecrosis by local ethanol injection can progress to early stage osteonecrosis. The different repair methods may have certain correlation with the results of osteonecrosis of the femoral heads.

To establish a method for screening nicotinamide phosphoribosyl transferase (NAMPT) inhibitors based on endogenous fluorescence determination.The double mutants of NAMPT, G355C/D393C, was cross-linked by using 1, 4-Bismaleimidobutane (BMB) to block the entrance of enzymatic active site of NAMPT. The binding of compounds to NAMPT was evaluated according to the change of spontaneous fluorescence of NAMPT and BMB-NAMPT with 280 nm excitation and 333 nm emmision. The in vitro enzamatic activity of NAMPT was determined by nuclear magnetic resonance. The cell viability was determined by MTT assay.FK866 significantly decreased the spontaneous fluorescence of NAMPT but not of BMB-NAMPT. Rosmaric, cynarine and 1, 3-dicaffeoylquinic acid also decreased the spontaneous fluorescence of both NAMPT and BMB-NAMPT. However, the inhibition on two proteins was equivalent. FK866 significantly inhibit the catalysis of NAMPT. Rosmarinic acid, cynarine and 1, 3-dicaffeoylquinic acid failed to inhibit the catalysis of NAMPT. FK866 inhibited the viability of A549 cells, but rosmarinic acid, cynarine and 1, 3-dicaffeoylquinic acid did not.Endogenous fluorescence spectrometry based on NAMPT and BMB-NAMPT protein can be used for screening compounds that bind with NAMPT, and distinguishing the binding site - either within the enzymatic active site or not. Rosmarinic acid, cynarine and 1, 3-dicoffeoylquinic acid can bind to NAMPT out its enzymatic active site.

To determine if combined therapy consisting of NEL-like type 1 gene (NELL-1) and zoledronate can prevent the collapse of the femoral head and stimulate the new bone formation in an animal model of osteonecrosis.Ischemic osteonecrosis was surgically induced in 24 SD rats, whicih were equally randomly divided into three groups: combination group, treated with both NELL-1 and zoledronate; sham operation group; and placebo group, treated with normal saline solution. The animals were killed 5 weeks after surgery. Radiography, MicroCT, histology, and immunohistochemistry were performed to analyze the results.Morphologically, the femoral head was at good shape in the combination group, while mildly flattened femoral head was seen in the placebo group. No heterotopic ossifications were observed in each group. MicroCT assessment showed significantly higher total and bone mineral volume in the combination group than in the placebo group (P0.01), whereas no such significant difference was found when compared with the sham operation group(P0.05). Histological assessment showed more active osteoblast activity and reduced osteoclast activity in the combination group compared with placebo group.A combination of NELL-1 and zoledronate can decrease the femoral head deformity while stimulating bone formation in a traumatic rat osteonecrois model, showing a potential to reverse the osteonecrosis.

To observe the effect and mechanism of zoledronate on prevention of collapse in an animal model of osteonecrosis.Ischemic osteonecrosis was surgically induced in 16 SD rats (which were further divided into zoledronate group and placebo group); another 8 rats were used as sham surgery group (n=8). The animals were killed 5 weeks after surgery. Radiographic, Micro-CT, histological, and immunohistochemical assessments were performed.Radiographic assessment showed better preservation of the femoral head shape in the zoledronate group than in the placebo group but not significantly different from the sham surgery group. Micro-CT assessment showed higher total volume, bone volume, and total mineralized content in the zoledronate group(all P0.05). Compared with the placebo group, the zoledronate group had reduced osteoclast and osteoblast activity, as confirmed by histological examinations.Zoledronate can decrease the femoral head deformity by reducing the osteoclast activity while suppressing new bone and vessels formation in a rat model of traumatic osteonecrosis, and therefore may delay the collapse of femoral head.

To analyze the reliability of magnetic resonance imaging (MRI) in detecting posterior ligament complex injury in thoracolumbar fractures.Ninety-five patients with thoracolumbar fracture were evaluated by palpation of the inter-spinal gap, plain radiography, and MRI before operation. In addition to conventional MRI sequences, a fat-suppressed T2-weighted sagittal sequence was performed. Surgery was performed by a posterior approach. During the operation, posterior ligament complex was examined.A wide inter-spinal gap was palpated in 41 patients and was found in 55 patients on plain radiography. According to MRI, injury to the supraspinal ligament was suspected in 85 patients, the inter-spinal ligament in 83 patients, and the ligamentum flavum in 26 patients. There were 82 supraspinal ligament injuries, 80 inter-spinal ligament injuries, and 20 ligamentum flavum injuries in operative findings. The relations between plain radiography and operative findings, between MRI interpretation and operative findings were statistically significant.A fat-suppressed T2-weighted sagittal sequence of MRI is a highly sensitive, specific, and accurate method to detect posterior ligament complex injury and which is recommended for the accurate evaluation of posterior ligament complex injury in thoracolumbar fractures.

To investigate the feasibility that transforming growth factor-beta1 (TGF-beta1) -loaded fibrin sealant (FS) promotes bone marrow mesenchymal stem cells (BMSCs) to create tissue engineering cartilage in vivo.The BMSCs were isolated from healthy human and amplified in vitro, and then induced by defined medium containing TGF-beta1 and dexamethasone. After 7 days the induced BMSCs were collected and mixed with TGF-beta1-loaded FS or FS as BMSCs+ FS-TGF-beta1 group and BMSCs+ FS experimental group. Then the mixture was injected by a needle into the dorsum of nude mice. In control group, only FS or BMSCs were injected. The tissue engineering specimens were harvested from nude mice 12 weeks later. Gross observation, average wet weight measurement, glycosaminoglycan (GAG) quantification, histology and immunohistochemistry were used to evaluate the results.The BMSCs have possessed the shape and functional characters of chondrocyte when transferred to a defined medium. After injection of the mixture, the cartilage-like tissue were formed in two experimental groups. Compared with BMSC+ FS group, the specimens of BMSCs +FS-TGF-beta1 group were larger and firmer. Alcian staining showed better metachromatic matrix formation. The GAG contents were significantly higher. Immunohistochemical staining of collagen type II was stronger. However, no cartilage-like tissue was formed in two control groups.TGF-beta1-loaded FS can promote BMSCs to contract injectable tissue engineering cartilage in vivo.