NLRC5 negatively regulates the NF-kappaB and type I interferon signaling pathways.

Stringent control of the NF-kappaB and type I interferon signaling pathways is critical to effective host immune responses, yet the molecular mechanisms that negatively regulate these pathways are poorly understood. Here, we show that NLRC5, a member of the highly conserved NOD-like protein family, can inhibit the IKK complex and RIG-I/MDA5 function. NLRC5 inhibited NF-kappaB-dependent responses by interacting with IKKalpha and IKKbeta and blocking their phosphorylation. It also interacted with RIG-I and MDA5, but not with MAVS, to inhibit RLR-mediated type I interferon responses. Consistent with these observations, NLRC5-specific siRNA knockdown not only enhanced the activation of NF-kappaB and its responsive genes, TNF-alpha and IL-6, but also promoted type I interferon signaling and antiviral immunity. Our findings identify NLRC5 as a negative regulator that blocks two central components of the NF-kappaB and type I interferon signaling pathways and suggest an important role for NLRC5 in homeostatic control of innate immunity.

A meta-analysis of the risk factors for lateral epicondylitis.

BACKGROUND: Very few meta-analyses discussed risk factors for lateral epicondylitis (LE), and previous meta-analyses reached conflicting conclusions with each other on some specific risk factors. PURPOSE: To investigate the risk factors for LE through meta-analysis. STUDY DESIGN: Meta-analysis. METHODS: PubMed, Embase, and Web of Science databases were searched for relevant studies in January 2022. Raw data were extracted into a predefined worksheet, and quality analysis was conducted by the Quality in Prognosis Studies (QUIPS) tool. Pooled effect sizes and 95% confidence intervals were calculated. R package "meta" was used for statistical analysis. RESULTS: 22 studies were included in the meta-analysis. Female sex (odds ratio [OR]=1.33 and p-value<0.05), smoking history (OR=1.46 and p-value<0.001), manual labor (OR=2.39 and p-value<0.001), and hypercholesterolemia (OR=1.67 and p-value<0.05) were significant risk factors for LE. CONCLUSIONS: Female gender, smoking history, manual labor, and hypercholesterolemia could increase the risk of LE. According to an additional literature review, statin treatment for hypercholesterolemia is described as potentially related to the development of LE.

Efficacy evaluation of chimeric antigen receptor-modified human peritoneal macrophages in the treatment of gastric cancer.

BACKGROUND: Gastric cancer is one of the most common cancers. Peritoneal carcinomatosis (PC) appears to be the most common pattern of recurrence, and more than half of the GC patients eventually die from PC. Novel strategies for the management of patients with PC are urgently needed. Recently, rapid progress has been made in adoptive transfer therapy by using macrophages as the effector cells due to their capabilities of phagocytosis, antigen presentation, and high penetration. Here, we generated a novel macrophage-based therapy and investigated anti-tumoral effects on GC and potential toxicity. METHODS: We developed a novel Chimeric Antigen Receptor-Macrophage (CAR-M) based on genetically modifying human peritoneal macrophages (PMs), expressing a HER2-FcεR1γ-CAR (HF-CAR). We tested HF-CAR macrophages in a variety of GC models in vitro and in vivo. RESULTS: HF-CAR-PMs specifically targeted HER2-expressed GC, and harboured the FcεR1γ moieties to trigger engulfment. Intraperitoneal administration of HF-CAR-PMs significantly facilitated the HER2-positive tumour regression in PC mouse model and prolonged the overall survival rate. In addition, the combined use of oxaliplatin and HF-CAR-PMs exhibited significantly augment anti-tumour activity and survival benefit. CONCLUSIONS: HF-CAR-PMs could represent an exciting therapeutic option for patients with HER2-positive GC cancer, which should be tested in carefully designed clinical trials.

Proteomic Landscape of Exosomes Reveals the Functional Contributions of CD151 in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Patients with TNBC have poor overall survival because of limited molecular therapeutic targets. Recently, exosomes have been recognized as key mediators in cancer progression, but the molecular components and function of TNBC-derived exosomes remain unknown. The main goal of this study was to reveal the proteomic landscape of serum exosomes derived from ten patients with TNBC and 17 healthy donors to identify potential therapeutic targets. Using a tandem mass tag-based quantitative proteomics approach, we characterized the proteomes of individual patient-derived serum exosomes, identified exosomal protein signatures specific to patients with TNBC, and filtered out differentially expressed proteins. Most importantly, we found that the tetraspanin CD151 expression levels in TNBC-derived serum exosomes were significantly higher than those exosomes from healthy subjects, and we validated our findings with samples from 16 additional donors. Furthermore, utilizing quantitative proteomics approach to reveal the proteomes of CD151-deleted exosomes and cells, we found that exosomal CD151 facilitated secretion of ribosomal proteins via exosomes while inhibiting exosome secretion of complement proteins. Moreover, we proved that CD151-deleted exosomes significantly decreased the migration and invasion of TNBC cells. This is the first comparative study of the proteomes of TNBC patient-derived and CD151-deleted exosomes. Our findings indicate that profiling of TNBC-derived exosomal proteins is a useful tool to extend our understanding of TNBC, and exosomal CD151 may be a potential therapeutic target for TNBC.

Mass cytometry reveals immune atlas of urothelial carcinoma.

Immunotherapy has emerged as a robust clinical strategy for cancer treatment. PD1/PD-L1 inhibitors have been used as second-line therapy for urothelial carcinoma due to the high tumor mutational burden. Despite the efficacy of the treatment is significant, the response rate is still poor. The tumor immune microenvironment plays a key role in the regulation of immunotherapeutic efficacy. However, a comprehensive understanding of the intricate microenvironment in clinical samples remains unclear. To obtain detailed systematic tumor immune profile, we performed an in-depth immunoassay on 12 human urothelial carcinoma tissue samples and 14 paratumor tissue samples using mass cytometry. Among the large number of cells assayed, we identified 71 T-cell phenotypes, 30 tumor-associated macrophage phenotypes. T cell marker expression profiles showed that almost all T cells in the tumor tissue were in a state of exhaustion. CD38 expression on tumor-associated macrophages (TAMs) was significantly higher than PDL1, and CD38+ TAMs were closely associated with immunosuppression. CD38 may be a more suitable target for immunotherapy in urothelial carcinoma compared to PD1/PDL1. This single-cell analysis of clinical samples expands our insights into the immune microenvironment of urothelial carcinoma and reveals potential biomarkers and targets for immunotherapy development.

Circ_0062582 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro by elevating SMAD5 expression through sponging miR-197-3p.

Circular RNAs (circRNAs) play crucial roles in many human diseases. However, the functions of circRNAs in osteoporosis (OP) are barely reported. In this study, we aimed to explore the function of circ_0062582 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. Circ_0062582 and SMAD5 were downregulated and miR-197-3p was upregulated in OP patients and increased in osteoblast medium (OM)-induced hBMSCs in vitro. Circ_0062582 knockdown inhibited the viability and osteogenic differentiation of hBMSCs. Circ_0062582 directly targeted miR-197-3p and miR-197-3p inhibition reversed the effects of circ_0062582 on hBMSC viability and osteogenic differentiation. SMAD5 was the target gene of miR-197-3p. SMAD5 overexpression promoted the viability and osteogenic differentiation of hBMSCs and attenuated miR-197-3p-mediated suppressive roles in hBMSC viability and osteogenic differentiation. In conclusion, circ_0062582 sponged miR-197-3p to elevate SMAD5 expression, thereby inducing hBMSC proliferation and osteogenic differentiation in vitro.

Microbial transformation and inhibitory effect assessment of uvaol derivates against LPS and HMGB1 induced NO production in RAW264.7 macrophages.

For the discovery of new pentacyclic triterpenes as a potential anti-inflammatory agent, microbial transformation of uvaol by Penicilium griseofulvum CICC 40293 and Streptomyces griseus ATCC 13273 was investigated. Stereoselective hydroxylation and epoxidation reactions were observed in the biotransformation. Moreover, six new metabolites were isolated and structurally elucidated by HR-ESI-MS and NMR spectrum. All the compounds were evaluated upon the inhibitory effects of nitric oxide (NO) release in RAW 264.7 cells induced by lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1). Among them, compound 3 (13, 28-epoxy-3ß, 7ß, 21ß-trihydroxy-urs-11-ene) with the unique epoxy structure and compound 5 (3ß, 21ß, 24, 28-tetrahydroxy-urs-12-en-30-oic acid), exhibited a considerable inhibitory effect on both models while compound 2 (urs-12-ene-3ß, 7ß, 21ß, 28-tetraol) showed a significant bias in the LPS-induced inflammatory response with IC50 value of 2.22 µM. Therefore, this study could provide some insights on the discovery of the pentacyclic triterpene leads for the treatment of either DAMPs or PAMPs triggered inflammation.

Microbial transformation of pentacyclic triterpenes for anti-inflammatory agents on the HMGB1 stimulated RAW 264.7 cells by Streptomyces olivaceus CICC 23628.

High mobility group box-1 protein (HMGB1) is a typical Damage-Associated Molecular Patterns (DAMPs) released in response to cellular inflammation. The pentacyclic triterpenes (PTs) are considered to be the natural inhibitors against HMGB1-related inflammation. To explore new lead compounds of PTs as anti-inflammatory agents, biotransformation of four PTs by Streptomyces olivaceus CICC 23628 was investigated in this study. As a result, thirteen unique 3,4-seco-triterpenes metabolites were isolated and twelve of them were first identified and reported. Structures of metabolites were determined based on HR-ESI-MS, 1D/2D NMR, and single-crystal X-ray diffraction. Furthermore, all compounds were subjected to the bioassay on the model of HMGB1-stimulated RAW 264.7 cells to evaluate their anti-inflammatory activity through nitric oxide (NO) inhibition activity. Compounds 3b (3,4-seco-olean-12-en-4,21α,22ß,24-tetrahydroxy-ol-3-oic acid) and 2b (3,4-seco-olean-12-en-4,21ß,22ß,24,29-pentahydroxy-ol-3-oic acid) exhibited NO inhibitory activity with IC50 values of 15.94 µM and 36.00 µM, respectively. Thus, indicating their potential as HMGB1 inhibitors and in developing potent anti-inflammatory agents. This work provides an operationally simple, efficient method for the rapid diversification of the PTs scaffold for a variety of distinctive 3,4-seco-triterpenes to facilitate the discovery of potential anti-inflammatory compounds.

Increased release of microvesicles containing mitochondria is associated with the myeloid differentiation of AML-M5 leukaemia cells.

Extensive literature has demonstrated that acute myeloid leukaemia (AML) cells show enhanced mitochondrial biogenesis and increased reliance on oxidative phosphorylation (OXPHOS) compared with normal hematopoietic progenitors, and one hallmark of AML leukaemia blasts is myeloid differentiation blockade. However, relatively few reports have linked these processes. Recent studies have indicated that therapies that overcome differentiation arrest represent an effective treatment strategy. Here, we identified that the disruption of the mitochondrial mass and energy metabolism promotes leukaemia cellular myeloid differentiation. In this study, we showed that acute monocytic leukaemia (AML-M5) cells package mitochondria in microvesicles (MVs) when MVs shed from membranes. Additionally, during myeloid differentiation, we report for the first time that differentiated leukaemia cells release more MVs than undifferentiated leukaemia cells. Targeting the formation of MVs using a specific inhibitor (Y-27632) restrained myeloid differentiation, suggesting that the increased release level of MVs plays an important role in regulating myeloid differentiation. Furthermore, the intracellular mitochondria and ATP levels were decreased after leukaemia cells overcame the differentiation blockade. Moreover, rotenone, which is used to inhibit the respiratory chain and ATP production, had a strong effect on myeloid differentiation in monocytic leukaemia cells. Collectively, these studies uncovered the relationship between mitochondrial function and myeloid differentiation and may provide more insight into the diagnosis and treatment of AML.

M2 polarization of tumor-associated macrophages is dependent on integrin ß3 via peroxisome proliferator-activated receptor-γ up-regulation in breast cancer.

Macrophages are particularly abundant and play an important role throughout the tumor progression process, namely, tumor-associated macrophages (TAM) in the tumor microenvironment. TAM can be polarized to disparate functional phenotypes, the M1 and M2 macrophages. M1-like type macrophages are defined as pro-inflammatory cells involved in killing cancer cells, while M2-like type cells can specially promote tumor growth and metastasis, tissue remodeling and immunosuppression. In this study, we first found that integrin ß3 was highly expressed on the surface of TAM, both in vivo and in vitro, that displayed the M2-like characteristics. Under intervention of CYC or triptolide, the integrin ß3 inhibitors, the M2 polarization of TAM could be inhibited. Moreover, in the cell model of M2 polarization, either blockade or knockout/knockdown of integrin ß3 could also suppress macrophage M2 polarization, which suggested that the M2 polarization was dependent on integrin ß3. Using knockdown of peroxisome proliferator-activated receptor-γ (PPARγ), an M2 regulator, we found that expression and activation of PPARγ participated in M2 polarization that was mediated by integrin ß3. Finally, to verify the activity of integrin ß3 inhibitors on TAM in vivo, 4T1 tumor-bearing mice were treated with CYC or triptolide; in response, the M1/M2 ratio of TAM was up-regulated, while the infiltration of total lymphocytes into tumor tissue was not altered. In general, our study found a connection between integrin ß3 and macrophage polarization, which provides a strategy for facilitating M2 to M1 repolarization and reconstructing the tumor immune microenvironment.

Expression and functions of cellular prion proteins in immunocytes.

Prion diseases are fatal neurodegenerative processes caused by the accumulation of the pathological prion protein, PrPSc . While pathological lesions are limited to the central nervous system (CNS), disease-specific proteins accumulate and replicate in secondary lymphoid organs prior to neuroinvasion, and their replication there depends on the abundance of cellular prion protein (PrPC ). PrPC is expressed in both central and peripheral lymphoid tissues, and up- or downregulates innate and adaptive immune responses. In addition to prion diseases, PrPC is also immunologically involved in other neurological disorders and infectious diseases, including Alzheimer's disease and human immunodeficiency virus infection. Herein, we summarize the expression and functions of PrPC in various immunocytes, as well as its immunological and pathological roles in neurodegeneration and infection.

Microbial transformation of glycyrrhetinic acid derivatives by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741.

Glycyrrhetinic acid (GA), the major bioactive pentacyclic triterpene aglycone of licorice root, was known to play a vital role in anti-ulcer, anti-depressant, anti-inflammatory, and anti-allergic. In this study, we semi-synthesized five GA derivatives by a series of chemical reactions. They were selected as substrates for the biotransformation and yielded thirteen metabolites by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741. Their structures were identified on the basis of extensive spectroscopic methods and nine of them were found for the first time. Two main types of reactions, regio- and stereo-selective hydroxylation and glycosylation, especially in the unactivated C-H bonds including C-11, C-19 and C-27, were observed in the biotransformation process, which greatly expand the chemical diversities of GA derivatives. All compounds were tested for their inhibitory effects on nitric oxide (NO) generation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Among them, olean-12-ene-3ß,7ß,15α,19α,30-pentol (16) and olean-12-ene-3ß,7ß,15α,27,30-pentol (17) showed significant inhibitory effect with IC50 values of 0.64 and 0.07 µM, respectively.

Biocatalytic allylic hydroxylation of unsaturated triterpenes and steroids by Bacillus megaterium CGMCC 1.1741.

In this study, we described the microbial catalyzed allylic oxidation by Bacillus megaterium CGMCC 1.1741 of three Δ12-pentacyclic triterpenes, erythrodiol (1), uvaol (2), hederagenin (3) and of four steroids including Δ5-steroids, diosgenin (4), pennogenin (5), 25(R,S)-ruscogenin (6) and Δ4-steroid, diosgenone (7). As a result, fourteen metabolites were generated with allyl hydroxyl moiety. Ten (1a-c, 2a, 2c, 3a, 5a-b, and 6a-b) of them were new natural products and their structures were determined on the basis of 1D/2D NMR and HR-MS data. Biocatalytic allylic oxidation by B. megaterium CGMCC 1.1741 is thus a potential non-toxic and efficient alternative method toward metal-mediated oxidation procedures in the synthesis of natural products and medicines.

Time Evolution Features of Entropy Generation Rate in Turbulent Rayleigh-Bénard Convection with Mixed Insulating and Conducting Boundary Conditions.

Time evolution features of kinetic and thermal entropy generation rates in turbulent Rayleigh-Bénard (RB) convection with mixed insulating and conducting boundary conditions at Ra = 109 are numerically investigated using the lattice Boltzmann method. The state of flow gradually develops from laminar flow to full turbulent thermal convection motion, and further evolves from full turbulent thermal convection to dissipation flow in the process of turbulent energy transfer. It was seen that the viscous, thermal, and total entropy generation rates gradually increase in wide range of t/τ < 32 with temporal evolution. However, the viscous, thermal, and total entropy generation rates evidently decrease at time t/τ = 64 compared to that of early time. The probability density function distributions, spatial-temporal features of the viscous, thermal, and total entropy generation rates in the closed system provide significant physical insight into the process of the energy injection, the kinetic energy, the kinetic energy transfer, the thermal energy transfer, the viscous dissipated flow and thermal dissipation.

Chimeric antigen receptor macrophage therapy for breast tumours mediated by targeting the tumour extracellular matrix.

BACKGROUND: The extracellular matrix (ECM) is essential for malignant tumour progression, as it is a physical barrier to various kinds of anticancer therapies. Matrix metalloproteinase (MMPs) can degrade almost all ECM components, and macrophages are an important source of MMPs. Studies using macrophages to treat tumours have shown that macrophages can enter tumour tissue to play a regulatory role. METHODS: We modified macrophages with a designed chimeric antigen receptor (CAR), which could be activated after recognition of the tumour antigen HER2 to trigger the internal signalling of CD147 and increase the expression of MMPs. RESULTS: Although CAR-147 macrophage treatment did not affect tumour cell growth in vitro compared with control treatment. However, we found that the infusion of CAR-147 macrophages significantly inhibited HER2-4T1 tumour growth in BALB/c mice. Further investigation showed that CAR-147 macrophages could reduce tumour collagen deposition and promote T-cell infiltration into tumours, which were consistent with expectations. Interestingly, the levels of the inflammatory cytokines TNF-α and IL-6, which are key factors in cytokine release syndrome, were significantly decreased in the peripheral blood in CAR-147 macrophage-transfused mice. CONCLUSION: Our data suggest that targeting the ECM by engineered macrophages would be an effective treatment strategy for solid tumours.

Is phosphorus a limiting factor to regulate the growth of phytoplankton in Daya Bay, northern South China Sea: a mesocosm experiment.

Previous field investigations implied a potential phosphorus (P)-limitation on the growth of phytoplankton in Daya Bay, a mesotrophic bay in the northern South China Sea. Using a total of 15 mesocosms (3 × 3 × 1.5 m, with ~10.8 m3 natural seawater containing phytoplankton assemblages for each), we found P-enrichment caused no obvious effect on phytoplankton (Chl a) growth across 8-day's cultivation in neither winter nor summer, while nitrogen (N)-enrichment greatly increased Chl a in both seasons. N plus P-enrichment further increased Chl a content. The N- or N plus P-enrichments increased the allocation of nano-Chl a but decreased micro-Chl a in most cases, with no obvious effect by P-alone. Coincided with nutrients effect on Chl a content, N- or N plus P-enrichments significantly enhanced the maximum photochemical quantum yield of Photosystem II (FV/FM) and maximum relative electron transport rate (rETRmax), but declined the non-photochemical quenching (NPQ), as well as the threshold for light saturation of electron transport (EK); again, P-enrichment had no significant effect. Moreover, the absorption cross section for PSII photochemistry (σPSII) and electron transport efficiency (α) increased due to N- or N plus P-enrichments, indicating the increased nutrients enhance the light utilization efficiency through promoting PSII light harvesting ability, and thus to enhance phytoplankton growth. Our findings indicate that N- or N plus P-enrichments rigorously fuel phytoplankton blooms regardless of N:P ratios, making a note of caution on the expected P-deficiency or P-limitation on the basis of Redfield N:P ratios in Daya Bay.

Restoration of CD3+CD56+ cell level improves skin lesions in severe psoriasis: A pilot clinical study of adoptive immunotherapy for patients with psoriasis using autologous cytokine-induced killer cells.

Psoriasis is a chronic inflammatory skin disorder mediated by the cells and molecules of both the innate and adaptive immune systems. Autologous cytokine-induced killer (CIK) cell infusion is considered an effective and safe cancer treatment and is licensed for this use in China. Accumulated evidence indicating that CD3+CD56+ cells are significantly decreased in psoriatic patients prompted us to investigate if the restoration of CD3+CD56+ cells may be beneficial for psoriatic patients. We designed a clinical trial for psoriasis treatment that involved CIK cell infusion because CIK cells include a large amount of CD3+CD56+ T cells (NCT01894373 at www.clinicaltrials.gov). Six patients with severe psoriasis were initially enrolled, and four of them exhibited markedly lower levels of CD3+CD56+ cells in their peripheral blood (PB) relative to healthy donors. CIK cell infusion-associated toxicity was not observed in any infusion. The percentage of CD3+CD56+ cells in the PB markedly increased and the psoriasis area and severity index (PASI) synchronously decreased in four patients with lower CD3+CD56+ cell contents, and two of them obtained a more than 4-month PASI75 after completing a four-cycle treatment. However, a decrease in the CD3+CD56+ cells was observed concomitantly with disease recurrence after short-term amelioration. In contrast, no obvious improvement was observed in the two patients with nearly normal CD3+CD56+ cells in the PB before treatment. These observations suggest that the normalization of the CD3+CD56+ cell level may improve the skin lesions of severe psoriasis and warrant further clinical trials for severe psoriasis using repeated CIK adoptive immunotherapy.

Identification of Caspase-6 as a New Regulator of Alternatively Activated Macrophages.

Alternatively activated macrophages (AAMs) play essential roles in the promotion of tissue remodeling, vasculogenesis, and tumor progression; however, the detailed mechanisms underlying the activation of AAMs remain largely unknown. Here, by using quantitative proteomic analysis, we identified 62 proteins that were up-regulated in IL-4-induced macrophages. Among these, Caspase-6 was increased significantly. Caspase-6 is important in the apoptotic signaling pathway; however, its role in non-apoptosis is also reported. Here, we first examined the non-apoptotic role of Caspase-6 in the alternative activation of macrophages after administration of IL-4, 4T1 tumor conditional medium, or co-culture with 4T1 cells. Both treatments promoted alternative activation of RAW264.7 cells and primary macrophages, whereas disruption of caspase-6 expression and activity could markedly suppress the biomarker levels of AAMs. Overexpression of Caspase-6 could significantly promote the activation of AAMs. Importantly, we further present evidence that caspase-6 could regulate breast cancer cell invasion by modulating MMP-2 and MMP-9 expression in 4T1 tumor-associated macrophages, as ablation of protein levels or activity of caspase-6 suppressed tumor cell invasion in vitro In conclusion, the observed results markedly expanded our views of the dynamic changes in protein composition during alternative activation of macrophages, and they revealed a critical new role of caspase-6 in regulating this cellular biological process, which suggested that caspase-6 might be a key nod molecule to regulate immunological steady-state and be a therapeutic candidate for tumor immunotherapy.

Cortical spreading depression preconditioning mediates neuroprotection against ischemic stroke by inducing AMP-activated protein kinase-dependent autophagy in a rat cerebral ischemic/reperfusion injury model.

Cortical spreading depression (CSD), based on its similarities with peri-infarct depolarization, is an ideal model for investigating transformation from the ischemic penumbra to infarct core. However, the underlying mechanisms remain unclear. To our knowledge, this is the first study to use a middle cerebral artery occlusion ischemic-reperfusion (I/R) injury model to determine whether AMP-activated protein kinase (AMPK)-dependent autophagy contributes to the neuroprotection of CSD preconditioning in rat cortex. In this study, we topically applied a pledget soaked in 1 mol/L KCl solution on rat cortex for 2 h to elicite CSD or 1 mol/L NaCl solution as a control. The results demonstrated that CSD preconditioning significantly decreased the infarct volume, neurological deficits and neuronal apoptosis in the cortical penumbra of middle cerebral artery occlusion rats, which was inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 200 nmol). Furthermore, CSD increased the protein levels of the autophagy markers LC3-II, Beclin-1 and the p-AMPK (Thr172 )/AMPK ratio at 12 h and decreased P62 and p-P70S6K (Thr389 ). Moreover, the AMPK inhibitor Compound C (20 mg/kg) down-regulated the LC3-II, p-AMPK (Thr172 )/AMPK and ULK1 levels, up-regulated the P62 and p-P70S6K (Thr389 ) levels induced by CSD. The neuroprotection of CSD is likely a result of AMPK-mediated autophagy activity and autophagy-induced neuronal cells apoptosis inhibition. These novel findings support a central role for AMPK and autophagy in CSD-induced ischemic tolerance. AMPK-mediated autophagy may represent a new target for stroke.

Soluble polymorphic bank vole prion proteins induced by co-expression of quiescin sulfhydryl oxidase in E. coli and their aggregation behaviors.

BACKGROUND: The infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrPC to PrPSc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrPC (BVPrP) is highly susceptible to PrPSc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease. RESULTS: To explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion. CONCLUSIONS: Our results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.