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Isoflurane (ISO) elicits protective effects on ischemia-induced brain injury. We investigated whether sub-anesthetic (0.7%) ISO post-conditioning attenuates the inflammation and apoptosis in oxygen-glucose deprivation (OGD)-insulted co-cultures (microglia and neurons) in vitro and the brain injury of the middle cerebral arterial occlusion (MCAO) rat. We demonstrated that ISO augmented the viability of OGD-treated microglia and neurons. ISO reduced the expression and activation of COX2 and iNOS in OGD-challenged microglia. ISO repressed the production of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 in OGD-exposed microglia. ISO also decreased nucleosomal fragmentation and caspase-3 activity but increased mitochondrial membrane potential in OGD-stimulated microglia and neurons. Mechanistically, ISO suppressed OGD-induced microglial inflammation by blocking ROS-regulated p38 MAPK/NF-κB signaling pathway and hampered OGD-triggered microglial apoptosis in a ROS- or NO-dependent fashion. In vivo results with MCAO rats were partly consistent with the in vitro observation. These findings indicate that sub-anesthetic ISO post-conditioning abates the inflammation and apoptosis in OGD-stimulated rat microglia and the apoptosis of OGD-exposed neurons and the brain injuries of MCAO rats, suggesting it as a potentially effective therapeutic approach for ischemic brain damages.

In this study, we investigated the role of ubiquitin-specific protease 22 (USP22) in the growth and progression of gastric cancer (GC). USP22 mRNA and protein levels were significantly higher in GC tissue samples and GC cell lines than in adjacent noncancerous tissue samples and a normal gastric mucosal epithelial cell line (GES1), respectively. USP22 knockdown significantly decreased in vitro survival, proliferation, migration, and invasiveness of GC cells compared with the controls. Western blot analysis of control and USP22-silenced GC cells showed that USP22 modulates the c-Myc/NAMPT/SIRT1-dependent FOXO1 and YAP signaling pathways. Subcutanenous injection of USP22-silenced GC cells into SCID mice generated significantly smaller xenograft tumors than did control cells. Moreover, USP22-silenced GC cells showed less lung metastasis than the controls following tail vein injection in SCID mice. In addition, high USP22 expression correlated positively with tumor size, advanced stage and metastasis, and correlated negatively with tumor differentiation and prognosis in GC patients. These results show that USP22 regulates growth and progression of GC via the c-Myc/NAMPT/SIRT1-dependent FOXO1 and YAP signaling pathways.

There is much interest in using chickens as “bioreactors” to produce large quantities of biopharmaceuticals. However, transient expression of foreign genes have been known to cause low efficiency of obtaining transgenic offspring, especially when using nonviral vectors. In present study, a transgenic chicken model was investigated to determine whether an exogenous gene can be expressed stably and transferred to its offspring through a matrix attachment region (MAR)-mediated non-viral vector using the eGFP marker gene. The eukaryotic expression vector pEGFP-N1-MAR, which contains the eGFP gene and MAR, was constructed and transfected into a chicken stage-X blastoderm to produce a G0 generation of transgenic chickens. The hatchabilities of different injection regions were tested; 18 of the 40 eggs injected with pEGFPN1- MAR in the area opaca hatched after 21 days of incubation, and had a hatchability rate of 45%. By contrast, eggs injected at the area pellucida did not hatch. Results from the fluorescence signal detection and polymerase chain reaction (PCR) verified that four hatched chicks from the G0 generation expressed the eGFP gene. Furthermore, fluorescence signal detection results indicated that 2 of the 65 chicks from the G1 generation expressed the eGFP gene. We conclude that MAR facilitates the production of transgenic chickens; pEGFP-N1-MAR application is a novel approach that can produce transgenic chicken offspring.

// Ling Qin 1,* , Zhi-qiang Yao 2,* , Qi Chang 3,* , Ya-li Zhao 2,* , Ning-ning Liu 2,* , Xiao-shan Zhu 2 , Qin-qin Liu 2 , Li-feng Wang 4 , An-gang Yang 5 , Chun-fang Gao 2 and Jun-tang Li 2,3,4,5 1 Department of Hematology, First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology, Luoyang, Henan, China 2 Centre of Inflammation and Cancer Research, 150th Central Hospital of PLA, Luoyang, Henan, China 3 Centre of Biomaterial and Biophysics Research, Institute of Training Medicine, 150th Central Hospital of PLA, Luoyang, Henan, China 4 State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi’an, Shaanxi, China 5 State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, Xi’an, Shaanxi, China * These authors have contributed equally to this work Correspondence to: Jun-tang Li, email: // Chun-fang Gao, email: // Ling Qin, email: // Keywords : chronic colitis, swimming, inflammation, oxidative stress, apoptosis, Immunology and Microbiology Section, Immune response, Immunity Received : March 28, 2016 Accepted : December 15, 2016 Published : December 21, 2016 Abstract Increasing evidence suggests that regular physical exercise suppresses chronic inflammation. However, the potential inhibitory effects of swimming on dextran sulfate sodium (DSS)-induced chronic colitis, and its underlying mechanisms, remain unclear. In this study, rats were orally administered DSS to induce chronic colitis, and subsequently treated with or without swimming exercise. A 7-week swimming program (1 or 1.5 hours per day, 5 days per week) ameliorated DSS-caused colon shortening, colon barrier disruption, spleen enlargement, serum LDH release, and reduction of body weight gain. Swimming for 1.5 hours per day afforded greater protection than 1 hour per day. Swimming ameliorated DSS-induced decrease in crypt depth, and increases in myeloperoxidase activity, infiltration of Ly6G + neutrophils and TNF-α- and IFN-γ-expressing CD3 + T cells, as well as fecal calprotectin and lactoferrin. Swimming inhibited pro-inflammatory cytokine and chemokine production and decreased the protein expression of phosphorylated nuclear factor-κB p65 and cyclooxygenase 2, whereas it elevated interleukin-10 levels. Swimming impeded the generation of reactive oxygen species, malondialdehyde, and nitric oxide; however, it boosted glutathione levels, total antioxidant capacity, and superoxide dismutase and glutathione peroxidase activities. Additionally, swimming decreased caspase-3 activity and expression of apoptosis-inducing factor, cytochrome c, Bax, and cleaved-caspase-3, but increased Bcl-2 levels. Overall, these results suggest that swimming exerts beneficial effects on DSS-induced chronic colitis by modulating inflammation, oxidative stress, and apoptosis.

Ubiquitin-specific protease 22 (USP22) aberrance has been implicated in several malignancies; however, whether USP22 plays a role in anaplastic thyroid carcinoma (ATC) remains unclear. Here, we report that USP22 expression is highly elevated in ATC tissues, which positively correlated with tumor size, extracapsular invasion, clinical stages, and poor prognosis of ATC patients. In vitro assays showed that USP22 depletion suppressed ATC cell survival and proliferation by decreasing Rb phosphorylation and cyclin D2, inactivating Akt, and simultaneously upregulating Rb; USP22 silencing restrained cell migration and invasion by inhibiting epithelial-mesenchymal transition; USP22 knockdown promoted mitochondrion- mediated and caspase-dependent apoptosis by upregulating Bax and Bid and promoting caspase-3 activation. Consistent with in vitro findings, downregulation of USP22 in ATC cells impeded tumor growth and lung metastasis in vivo. These results raise the applicability for USP22 as a useful predictor of ATC prognosis and a potential therapeutic target for ATC.

// Jun-Tang Li 1,2,3,* , Wei-Qi Wang 4,5,6,* , Ling Wang 7,* , Ning-Ning Liu 1,* , Ya-Li Zhao 1,* , Xiao-Shan Zhu 1 , Qin-Qin Liu 1 , Chun-Fang Gao 1 , An-Gang Yang 2 and Lin-Tao Jia 3 1 Centre of Inflammation and Cancer Research, 150th Central Hospital of PLA, Luoyang, Henan, China 2 State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, Xi’an, Shaanxi, China 3 State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi’an, Shaanxi, China 4 State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, China 5 National Clinical Research Center for Oral Diseases, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, China 6 Shaanxi Clinical Research Center for Oral Diseases, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, China 7 Department of Anesthesiology, 150th Central Hospital of PLA, Luoyang, Henan, China * These authors have contributed equally to this work Correspondence to: Jun-Tang Li, email: // An-Gang Yang, email: // Lin-Tao Jia, email: // Keywords : isoflurane, zymosan, neutrophil, NMDA receptor, TLR2, Immunology and Microbiology Section, Immune response, Immunity Received : September 07, 2015 Accepted : April 18, 2016 Published : April 28, 2016 Abstract Neutrophil release of NO/ONOO – induces endothelial cell barrier dysfunction in inflammatory acute lung injury (ALI). Previous studies using zymosan-triggered inflammation and ALI model revealed that zymosan promotes inducible NO synthase (iNOS) expression in neutrophils, and that isoflurane inhibits zymosan-induced oxidative stress and iNOS biosynthesis. However, the underlying mechanisms remain largely unknown. We found here that in zymosan-primed neutrophils, iNOS is transcriptionally activated by NF-κB, whose nuclear translocation is triggered by excessive reactive oxygen species (ROS) and consequently activated p38 MAPK. ROS production is attributed to zymosan-initiated Toll-like receptor 2 (TLR2) signaling, in which the adaptor MyD88 recruits and activates c-Src, and c-Src activates NADPH oxidase to generate ROS. Subanesthetic isoflurane counteracts the aforementioned zymosan-induced signaling by targeting N-methyl-D-aspartic acid (NMDA) glutamate receptor and thereby suppressing calcium influx and c-Src activation. Whereas iNOS accelerates NO/ONOO – production in neutrophils which eventually promote protein leak from pulmonary microvascular endothelial cells (PMVEC), isoflurane reduced NO/ONOO – release from zymosan-treated neutrophils, and thus relieves trans-PMVEC protein leak. This study provides novel insights into the roles of neutrophils and the underlying mechanisms in zymosan-induced ALI, and has implications for the therapeutic potential of subanesthetic isoflurane in attenuating inflammatory responses causing lung endothelial cell damage.

Eukaryotic elongation factor-2 kinase (eEF2K), a unique calcium/calmodulin-dependent protein kinase, is well known to regulate apoptosis, autophagy and ER stress in many types of human cancers. Therefore, eEF2K would be regarded as a promising therapeutic target; however, the eEF2K-regulated mechanism and its targeted inhibitor still remain to be discovered in cancer. Herein, we constructed a protein-protein interaction (PPI) network of eEF2K and achieved an eEF2K-regulated ER stress subnetwork by bioinformatics prediction. Then, we found that the differential protein expressions involved in ER stress in the context of si-eEF2K-treated MCF-7 and MDA-MB-436 cells by iTRAQ-based analyses, respectively. Integrated into these aforementioned results, we constructed a core eEF2K-regulated ER stress subnetwork in breast cancer cells. Subsequently, we screened a series of candidate compounds targeting eEF2K and discovered a novel eEF2K inhibitor (cefatrizine) with an anti-proliferative activity toward breast cancer cells. Moreover, we found that cefatrizine induced ER stress in both MCF-7 and MDA-MB-436 cells. Interestingly, we demonstrated that the mechanism of cefatrizine-induced ER stress was in good agreement with our bioinformatics and proteomics-based results. In conclusion, these results demonstrate that a novel eEF2K inhibitor (cefatrizine) induces ER stress in breast cancer cells by integrating bioinformatics prediction, proteomics analyses and experimental validation, which would provide a clue for exploring more mechanisms of eEF2K and its targeted inhibitors in cancer therapy.

Whereas miR-101 is involved in the development and progression of breast cancer, the underlying molecular mechanisms remain to be elucidated. Here, we report that miR-101 expression is inversely correlated with the clinical stage, lymph node metastasis and prognosis in breast cancers. Introduction of miR-101 inhibited breast cancer cell proliferation and invasion in vitro and suppressed tumor growth and lung metastasis of in vivo. CX chemokine receptor 7 (CXCR7) is a direct target of miR-101, positively correlating with the histological grade and the incidence of lymph node metastasis in breast cancer patients. The effects of miR-101 were mimicked and counteracted by CXCR7 depletion and overexpression, respectively. STAT3 signaling downstream of CXCR7 is involved in miR-101 regulation of breast cancer cell behaviors. These findings have implications for the potential application of miR-101 in breast cancer treatment.

Objective Despite major advances in its diagnosis and treatment, gastric cancer (GC) remains a major life-threatening disease. Treatment of the disease is further aggravated by the lack of diagnostic biomarkers that can aid in the early detection of GC and promote its favorable prognosis. The present work aims to identify novel diagnostic biomarkers for GC. Design and methods The present work is a case–control study that focuses on proteomic analysis of serum from healthy volunteers and GC patients using ClinProt profiling technology based on mass spectrometry. A pattern of proteins/peptides with the ability to differentiate the studied populations was identified. Deregulated proteins/peptides differentially expressed in the serum of patients compared with healthy volunteers were identified by mass spectroscopy. Results A pattern of proteins/peptides consisting of four protein/peptide peaks at m/z 1467, 1867, 2701, and 2094 was identified. These protein/peptide peaks were able to differentiate the studied populations with close to 100% sensitivity and specificity. Three of the deregulated proteins/peptides at m/z 1867, 2701, and 2094 were identified by mass spectroscopy (LTQ Orbitrap XL) as tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b–c1 complex subunit 1, respectively. Conclusions The pattern of proteins/peptides identified in the present work shows great potential for GC diagnosis. Deregulated proteins of tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b–c1 complex subunit 1 may be involved in the pathogenesis of GC and serve as potential serological diagnostic biomarkers.

We investigated the effect of 1.4% isoflurane (ISO) on the development of inflammation and apoptosis caused by zymosan (ZY) in mice. We found that ZY-challenged mice exhibited significant body weight loss, markedly high mortality, and significant lung injury characterized by the deterioration of histopathology, histologic scores, and wet-to-dry ratio after ISO treatment. ISO dramatically attenuated ZY-induced lung neutrophil recruitment and inflammation, as evidenced by the reduced levels of total cells, neutrophils, and proinflammatory cytokines (i.e., tumor necrosis factor-α, interleukin- (IL-) 1β, IL-6, and macrophage inflammatory protein-2) in bronchoalveolar lavage fluid and of their mRNA expression in lung tissues. ISO also inhibited ZY-induced expression and activation of nuclear factor-kappaB p65 and inducible nitric oxide synthase in pulmonary tissue. ZY administration also resulted in the upregulation of heme oxygenase-1 expression and activity in the lung, which was further enhanced by ISO treatment. Moreover, ISO markedly prevented ZY-induced pulmonary cell apoptosis in mice, as reflected by the decrease in expression of procaspase-8, procaspase-3, cleaved caspase-8, and cleaved caspase-3, as well as in caspase-3 activity and Bcl-2-associated X/B-cell lymphoma 2 ratio. These results indicate that ISO is a potential therapeutic drug for treating ZY-induced lung injury, and further investigations are warranted.

Introduction The onset of distal metastasis, which underlies the high mortality of breast cancers, warrants substantial studies to depict its molecular basis. Nuclear factor of activated T cells 5 (NFAT5) is upregulated in various malignancies and is critically involved in migration and invasion of neoplastic cells. Nevertheless, the metastasis-related events potentiated by this transcriptional factor and the mechanism responsible for NFAT5 elevation in carcinoma cells remain to be fully elucidated. Methods The correlation of NFAT5 with breast cancer invasiveness was investigated in vitro and clinically. The genes transcriptionally activated by NFAT5 were probed and their roles in breast cancer progression were dissected. The upstream regulators of NFAT5 were studied with particular attempt to explore the involvement of non-coding RNAs, and the mechanism underlying the maintenance of NFAT5 expression was deciphered. Results In metastatic breast cancers, NFAT5 promotes epithelial-mesenchymal transition (EMT) and invasion of cells by switching on the expression of the calcium binding protein S100A4, and facilitates the angiogenesis of breast epithelial cells and thus the development of metastases by transcriptionally activating vascular endothelial growth factor C (VEGF-C). NFAT5 is directly targeted by miR-568, which is in turn suppressed by the long non-coding RNA, Hotair, via a documented in trans gene silencing pattern, that is recruitment of the polycomb complex (Polycomb Repressive Complex 2; PRC2) and LSD1, and consequently methylation of histone H3K27 and demethylation of H3K4 on the miR-568 loci. Conclusion This study unravels a detailed role of NFAT5 in mediating metastatic signaling, and provides broad insights into the involvement of Hotair, in particular, by transcriptionally regulating the expression of microRNA(s), in the metastasis of breast cancers. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0454-2) contains supplementary material, which is available to authorized users.

CD4+ T cells play critical roles in orchestrating adaptive immune responses. Their activation and proliferation are critical steps that occur before they execute their biological functions. Despite the important role of this process, the underlying molecular events are not fully understood. MicroRNAs (miRNAs) have been shown to play important roles in lymphocyte development and function. However, the miRNAs that regulate T-cell differentiation, activation and proliferation are still largely unknown. In our previous study, using a miRNA array, we found that several miRNAs (including miR-202, 33b, 181c, 568 and 576) are differentially expressed between resting and activated CD4+ T cells. In this study, we focused on the function of miR-568 during CD4+ T-cell activation. We showed that the expression level of miR-568 decreased during the activation of T cells, including Jurkat cells and human peripheral blood CD4+ T cells. When Jurkat or human peripheral blood CD4+ T cells were transfected with miR-568 mimics, cell activation was significantly inhibited, as shown by the inhibited expression of activation markers such as CD25, CD69 and CD154; decreased IL-2 production; and inhibited cell proliferation. Using software predictions and confirmatory experiments, we demonstrated that nuclear factor of activated T cells 5 (NFAT5) is a target of miR-568. Treg cells are an important CD4+ T-cell subpopulation, so we also evaluated the function of miR-568 in Treg-cell activation and differentiation. We showed that the miR-568 level decreased, while the NFAT5 protein level increased during CD4+CD25+ Treg-cell activation, and the transfection of miR-568 mimics inhibited the NFAT5 expression, inhibited the production of both TGF-β and IL-10 and also inhibited the proliferation of Treg cells. Our further study showed that over-expression of miR-568 can inhibit Treg-cell differentiation and can inhibit the suppressive effect of these cells on effector cells. In addition, inhibition of NFAT5 by siRNA-mediated knockdown can inhibit the activation and differentiation of Treg cells. These findings reveal that miR-568 can inhibit the activation and function of both CD4+ T cells and Treg cells by targeting NFAT5. Since miR-568 plays an important role in both CD4+ T cells and Treg cells, these findings may provide leads for the development of novel treatments for human inflammatory and autoimmune diseases.

Despite major advances in its diagnosis and treatment, gastric cancer (GC) remains a major life-threatening disease. Treatment of the disease is further aggravated by the lack of diagnostic biomarkers that can aid in the early detection of GC and promote its favorable prognosis. The present work aims to identify novel diagnostic biomarkers for GC.The present work is a case-control study that focuses on proteomic analysis of serum from healthy volunteers and GC patients using ClinProt profiling technology based on mass spectrometry. A pattern of proteins/peptides with the ability to differentiate the studied populations was identified. Deregulated proteins/peptides differentially expressed in the serum of patients compared with healthy volunteers were identified by mass spectroscopy.A pattern of proteins/peptides consisting of four protein/peptide peaks at m/z 1467, 1867, 2701, and 2094 was identified. These protein/peptide peaks were able to differentiate the studied populations with close to 100% sensitivity and specificity. Three of the deregulated proteins/peptides at m/z 1867, 2701, and 2094 were identified by mass spectroscopy (LTQ Orbitrap XL) as tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c₁ complex subunit 1, respectively.The pattern of proteins/peptides identified in the present work shows great potential for GC diagnosis. Deregulated proteins of tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c₁ complex subunit 1 may be involved in the pathogenesis of GC and serve as potential serological diagnostic biomarkers.

Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell's plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.) into the cytoplasm provides the proteases with access to numerous protein substrates that promote apoptosis after cleavage. These proteases are believed to be the main executioners of target cell apoptosis. Although the PRF and granzyme components are both critical to this process and in some way involved in inducing cell death in target cells, the inhibition of tumor growth could still be efficient in granzyme-deficient mice. It is unclear whether PRF alone can suppress tumors. In this study, we discovered that forced ectopic expression of PRF alone, in the absence of granzymes, could mediate cell death in cancer cells. Notably, transient expression of both full-length and truncated active-form PRF in human Hep G2, SK-BR-3, and HeLa cells was found to induce apparent cell growth inhibition and cell death, as evidenced by chromosome condensation and DNA fragmentation, increased caspase-3 activity, and the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. This PRF-induced cell death could be abrogated by pan-caspase inhibitor (Z-VAD) and mitochondria protector (TAT-BH4). The implication of these results is that ectopically expressed PRF has apoptosis-inducing abilities, and PRF alone is sufficient to induce apoptotic cell death in cells with ectopic expression. Taking this into consideration, our results suggest the possibility of using PRF as a pro-apoptotic gene for tumor therapeutics.

Multiple organ dysfunction syndrome (MODS) is one of the leading causes of death in the intensive care unit. Organ failure especially lung injury is highly associated with the mortality for MODS patients. Volatile anesthetic isoflurane (ISO) is one of the most widely used anesthetic agents, and ISO anesthesia has been reported to improve the survival rate and organ function in sepsis/MODS models. However, the application of anesthetic dose ISO in critically ill patients is limited. Compared with i.v. anesthetic pentobarbital treatment, we showed that twice inhalation of ISO at subanesthetic dose (0.7%, 0.5 minimum alveolar concentration) alleviated lung injury at 24 h after zymosan (ZY) injection and increased the 7-day survival rate from 10% to 45% in mice. We also showed that ISO exerted its protection by significantly improving the activities of superoxide dismutase and catalase in lung and serum when compared with those in pentobarbital-treated mice. The catalase inhibitor 3-amino-1, 2, 4-triazole partially abolished the protective effect of ISO in ZY-challenged mice. We conclude that subanesthetic dose ISO protects against ZY-induced generalized inflammation and its associated lung injury via enhancing the activities of antioxidant enzymes in mice, which may provide a new strategy for the treatment of critically ill patients.

A new control method for active power filters using support vector machine (SVM) is presented. In the strategy, SVM is employed to model and predict future harmonic compensating current, it has the advantages of nonexistence of local minima solutions, automatic choice of model complexity and good generalization performance. Based on the model output, Genetic algorithm optimization method is adopted to produce proper value of control vector, this value is adequately modulated by means of a space vector PWM modulator which generate proper gating patterns of the inverter switches to maintain tracking of reference current. The SVM based predictive algorithm is used in internal model control scheme to compensate for process disturbances, measurement noise and modeling errors. The proposed method is applied to the control of a shunt active power filter, simulation results show SVM based predictive controller is more effective and feasible than PI control or digit adaptive control.