Urotensin II (UII) and its receptor (UTR) have mitogenic effects on tumor growth. Our previous study demonstrated that the UII/UTR system is up-regulated in dithyinitrosamine-induced precancerous rat liver lesions. However, its role in human hepatocellular carcinoma remains unknown. In this study, the mRNA and protein expression of UII and its receptor (UTR) in human hepatocellular carcinoma samples and in the BEL-7402 human hepatoma cell line were evaluated. In addition, the effect of exogenous UII on the pathways that regulate proliferation in BEL-7402 cells in vitro were determined. Liver sections were subjected to immunohistochemical staining. mRNA expression was detected by real-time polymerase chain reaction analysis, and protein levels were evaluated by western blotting. Proliferating cells were detected by BrdU incorporation. The expression of UII/UT mRNA and protein significantly increased in human hepatocellular carcinoma samples, and in BEL-7402 cells. Administration with UII increased the phosphorylation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK). Furthermore, GF109203x, PD184352, and SB203580 partially abolished UII-induced proliferation of BEL-7402 cells. These results provide the first evidence that up-regulation of the UII/UT system may enhance proliferation of the human hepatoma cell line at least in part via PKC, ERK1/2, and p38 MAPK signaling pathways, and may provide novel therapeutic targets for inhibiting human hepatocellular carcinoma.

A long-term "memory" of hyperglycemic stress, even when glycemia is normalized, has been previously reported in diabetes. In this report we propose a similar hypothesis that exposure to continuous high angiotensin II (Ang II) results in a cellular "memory" in isolated cardiomyocytes and in the heart tissues, and we investigate the role of NADPH oxidases in this phenomenon. Continuous high Ang II for 3 days markedly increased cardiomyocyte size, TUNEL-positive apoptotic cardiomyocytes, expression of inflammatory cytokines, and oxidative stress. These deleterious effects were also observed in the memory condition (high Ang II for 2 days followed by normal medium for 1 day). Furthermore, in a mouse model, Ang II infusion for 3 weeks significantly increased cardiac hypertrophy, apoptosis, inflammation, and ROS generation but decreased cardiac function compared with control mice, and similar effects were also observed in mice in the memory condition. Importantly, blockade of NADPH oxidase using apocynin diminished the induction of high Ang II stress markers in isolated cardiomyocytes and in the mouse heart. These effects were associated with inhibition of NADPH oxidase-mediated AKT/mTOR/S6K and ERK signaling pathways. The present results demonstrate the hypothesis that exposure to continuous high Ang II results in a hypertensive cellular memory that remains, even when cells or mice are switched back to normal Ang II. This phenomenon was associated with NADPH oxidase-mediated oxidative stress.

Somatic cells can be directly converted into functional neurons by ectopic expression of defined factors and/or microRNAs. Since the first report of conversion mouse embryonic fibroblasts into functional neurons, the postnatal mouse, and human fibroblasts, astroglia, hepatocytes, and pericyte-derived cells have been converted into functional dopaminergic and motor neurons both in vitro and in vivo. However, it is invasive to get all these materials. In the current study, we provide a noninvasive approach to obtain directly reprogrammed functional neurons by overexpression of the transcription factors Ascl1, Brn2, NeuroD, c-Myc, and Myt1l in human urine cells. These induced neuronal (iN) cells could express multiple neuron-specific proteins and generate action potentials. Moreover, urine cells from Wilson's disease (WD) patient could also be directly converted into neurons. In conclusion, generation of iN cells from nonneural lineages is a feasible and befitting approach for neurological disease modeling.

Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.

Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance.

Glioma is the most prevalent and fatal primary tumor of the central nervous system in adults, while the development of effective therapeutic strategies in clinical practice remain a challenge. Nucleotide-binding domain leucine-rich family pyrin-containing 3 (NLRP3) has been reported to be associated with tumorigenesis and progression; however, its expression and function in human glioma remain unclear. The present study was designed to explore the biological role and potential mechanism of NLRP3 in human glioma. The results demonstrated that overexpression of NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), caspase‑1 and interleukin (IL)‑1β protein in human glioma tissues were significantly correlated with higher World Health Organization grades. The in vitro biological experiments demonstrated that NLRP3 downregulation significantly inhibited the proliferation, migration and invasion, and promoted the apoptosis of SHG44 and A172 glioma cell lines. Furthermore, western blot assays revealed that the downregulation of NLRP3 significantly reduced the expression of ASC, caspase‑1 and IL‑1β protein. Furthermore, NLRP3 knockdown caused the inhibition of epithelial-mesenchymal transition (EMT), and inhibited the phosphorylation of AKT serine/threonine kinase (AKT) and phosphorylation of phosphatase and tensin homolog (PTEN). Consistently, the upregulation of NLRP3 significantly increased the expression of ASC, caspase‑1, IL‑1β and phosphorylated-PTEN, promoted proliferation, migration, invasion and EMT, inhibited apoptosis, and activated the AKT signaling pathway. The data of the present study indicate that NLRP3 affects human glioma progression and metastasis through multiple pathways, including EMT and PTEN/AKT signaling pathway regulation, enhanced inflammasome activation, and undefined inflammasome-independent mechanisms. Understanding the biological effects of NLRP3 in human glioma and the underlying mechanisms may offer novel insights for the development of glioma clinical therapeutic strategies.

Cancer patients frequently develop autoantibodies. To test the hypothesis that the appearance of autoantibodies precedes the clinical diagnosis of cancer, we applied an immunoproteomic approach to compare autoantibody profiles before and after appearance of malignances. Proteins from A549 cells, a lung adenocarcinoma cell line, were separated by two dimensional electrophoresis and then immunoblotted with serum samples from 8 individuals who were eventually diagnosed with lung cancer. Compared with autoantibody profiles from 3 years prior to the appearance of malignances, 21 immunoreactive spots newly appeared or presented with stronger staining intensity when clinical diagnoses were made. Among them, 10 matched spots on 2-DE gels were identified by mass spectrometry analysis as 5 proteins. With immunoprecipitation analysis, the antigenicity of protein cathepsin D was confirmed, and notably, in lung cancer sera, the occurrences of autoantibodies against the specific forms of cathepsin D differed significantly from the control groups (p<0.05). Our findings suggest that harnessing immunity may have utility for early cancer marker discovery, and that comparing autoantibodies to specific forms of cathepsin D may be a promising early marker of lung cancer.

Pressure overload-induced hypertrophic remodeling is a critical pathological process leading to heart failure (HF). Suppressor of cytokine signaling-3 (SOCS3) has been demonstrated to protect against cardiac hypertrophy and dysfunction, but its mechanisms are largely unknown. Using primary cardiomyocytes and cardiac-specific SOCS3 knockout (SOCS3cko) or overexpression mice, we demonstrated that modulation of SOCS3 level influenced cardiomyocyte hypertrophy, apoptosis and cardiac dysfunction induced by hypertrophic stimuli. We found that glucose regulatory protein 78 (GRP78) was a direct target of SOCS3, and that overexpression of SOCS3 inhibited cardiomyocyte hypertrophy and apoptosis through promoting proteasomal degradation of GRP78, thereby inhibiting activation of endoplasmic reticulum (ER) stress and mitophagy in the heart. Thus, our results uncover SOCS3-GRP78-mediated ER stress as a novel mechanism in the transition from cardiac hypertrophy to HF induced by sustained pressure overload, and suggest that modulating this pathway may provide a new therapeutic approach for hypertrophic heart diseases.

Urotensin II (UII) and the urotensin II receptor (UT) exhibit mitogenic effects on tumor growth. Our previous study demonstrated that the UII/UT system is upregulated in hepatocellular carcinoma (HCC) and may enhance the proliferation of human hepatoma cells. However, the clinical significance of UII/UT expression in HCC remains unclear. The present study assessed UII messenger RNA (mRNA) expression in 129 surgical specimens obtained from HCC patients using reverse transcription quantitative-polymerase chain reaction. The association between UII mRNA expression and clinicopathological parameters and overall survival rates was also investigated. The results revealed that UII and UT mRNA expression was significantly increased in HCC tissue compared with adjacent non-cancerous liver tissue (P<0.001). Furthermore, a significant correlation was identified between UII expression and histological differentiation (P<0.01), tumor size (P<0.01) and tumor stage (P=0.026). Kaplan-Meier survival analysis indicated that overall survival time was significantly shorter in patients with high UII expression, compared with those with low UII expression (P<0.001). Multivariate analyses indicated that UII expression was an independent predictor of overall survival (odds ratio, 1.12; P<0.001). In addition, UII mRNA was correlated with vascular endothelial growth factor mRNA expression. Therefore, UII expression is an independent biomarker for the prognosis of patients with HCC and thus, the UII/UT system may present a novel therapeutic target for the treatment of HCC.

Paroxysmal kinesigenic dyskinesia (PKD) is the most common subtype of paroxysmal dyskinesias and is caused by mutations in PRRT2 gene. The majority of familial PKD was identified to harbor PRRT2 mutations. However, over two-third of sporadic PKD patients did not carry anyPRRT2 mutation, suggesting an existence of additional genetic mutations or possible misdiagnosis due to clinical overlap.

Carboxyl terminus of Hsp70-interacting protein (CHIP) is a critical ubiquitin ligase/cochaperone to reduce cardiac oxidative stress, inflammation, cardiomyocyte apoptosis and autophage etc. However, it is unclear whether overexpression of CHIP in the heart would exert protective effects against DOX-induced cardiomyopathy. Cardiac-specific CHIP transgenic (CHIP-TG) mice and the wild-type (WT) littermates were treated with DOX or saline. DOX-induced cardiac atrophy, dysfunction, inflammation, oxidative stress and cardiomyocyte apoptosis were significantly attenuated in CHIP-TG mice. CHIP-TG mice also showed higher survival rate than that of WT mice (40% versus 10%) after 10-day administration of DOX. In contrast, knockdown of CHIP by siRNA in vitro further enhanced DOX-induced cardiotoxic effects. Global gene microarray assay revealed that after DOX-treatment, differentially expressed genes between WT and CHIP-TG mice were mainly involved in apoptosis, atrophy, immune/inflammation and oxidative stress. Mechanistically, CHIP directly promotes ubiquitin-mediated degradation of p53 and SHP-1, which results in activation of ERK1/2 and STAT3 pathways thereby ameliorating DOX-induced cardiac toxicity.

The emergence of mass spectrometry (MS)-based signatures as biomarkers has generated considerable enthusiasm among oncologists. However, variations in normal individuals also exist, and a better understanding of serum peptide patterns of healthy individuals will be important for further exploring disease-specific serum peptide patterns. Following development of a serum peptide pattern platform, we analyzed 500 serum samples obtained from healthy individuals. Samples from breast (n = 84), lung (n = 70), and rectal (n = 30) cancer patients were also examined. Extensive data analysis revealed negligible contributions of age to serum peptide patterns except in healthy individuals between 20-30 and 60+ years of age. Gender-related variations in the serum patterns of healthy individuals were only observed in 20-30 year-old individuals. Our results revealed substantial variation in individual peptide profiles, but 65 peptides were detected at a 20% higher frequency in the healthy population. A peptide profile was developed for each type of cancer, containing 10 discriminating peptides not prevalent in healthy individuals. Sequence identification of 111 signature peptides revealed that they fell into several tight clusters and most were exopeptidase products of serum resident proteins. We have obtained a MS-based serum peptide profile for healthy individuals, providing a reference for observing the occurrence of cancer-specific peptides.

Embryonic stem (ES) cells are pluripotent cells that are capable of giving rise to any type of cells in the body and possess unlimited self-renewal potential. However, the exact regulatory mechanisms that govern the self-renewal ability of ES cells remain elusive. To understand the immediate early events during ES cell differentiation, we performed a proteomics study and analyzed the proteomic difference in murine ES cells before and after a 6-h spontaneous differentiation. We found that the expression level of glutathione peroxidase-1 (GPx-1), an antioxidant enzyme, is dramatically decreased upon the differentiation. Both knockdown of GPx-1 expression with shRNA and inhibiting GPx-1 activity by inhibitor led to the differentiation of ES cells. Furthermore, we showed that during early differentiation, the quick degradation of GPx-1 was mediated by proteasome. Thus, our data indicated that GPx-1 is a key regulator of self-renewal of murine embryonic stem cells.

The N-terminal protein processing mechanism (NPM) including N-terminal Met excision (NME) and N-terminal acetylation (N(α)-acetylation) represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown.

Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.

Hypertensive cardiac remodeling is a major cause of heart failure. The immunoproteasome is an inducible form of the proteasome and its catalytic subunit β5i (also named LMP7) is involved in angiotensin II-induced atrial fibrillation; however, its role in deoxycorticosterone-acetate (DOCA)-salt-induced cardiac remodeling remains unclear. C57BL/6 J wild-type (WT) and β5i knockout (β5i KO) mice were subjected to uninephrectomy (sham) and DOCA-salt treatment for three weeks. Cardiac function, fibrosis, and inflammation were evaluated by echocardiography and histological analysis. Protein and gene expression levels were analyzed by quantitative real-time PCR and immunoblotting. Our results showed that after 21 days of DOCA-salt treatment, β5i expression and chymotrypsin-like activity were the most significantly increased factors in the heart compared with the sham control. Moreover, DOCA-salt-induced elevation of blood pressure, adverse cardiac function, chamber and myocyte hypertrophy, interstitial fibrosis, oxidative stress, and inflammation were markedly attenuated in β5i KO mice. These findings were verified in β5i inhibitor PR-957-treated mice. Moreover, blocking of PTEN (the gene of phosphate and tensin homolog deleted on chromosome ten) markedly attenuated the inhibitory effect of β5i knockout on DOCA-salt-induced cardiac remodeling. Mechanistically, DOCA-salt stress upregulated the expression of β5i, which promoted the degradation of PTEN and the activation of downstream signals (AKT/mTOR, TGF-β1/Smad2/3, NOX, and NF-κB), which ultimately led to cardiac hypertrophic remodeling. This study provides new evidence of the critical role of β5i in DOCA-salt-induced cardiac remodeling through the regulation of PTEN stability, and indicates that the inhibition of β5i may be a promising therapeutic target for the treatment of hypertensive heart diseases.

Cardiac hypertrophy without appropriate treatment eventually progresses to heart failure. Our recent data demonstrated that the immunoproteasome subunit β5i promotes cardiac hypertrophy. However, whether β5i is a promising therapeutic target for treating hypertrophic remodeling remains unknown. Here, we investigated the effects of PR-957, a β5i-specific inhibitor, on angiotensin II (Ang II)-induced hypertrophic remodeling in the murine heart. The infusion of Ang II increased immunoproteasome chymotrypsin-like activity and β5i catalytic subunit expression in the heart, whereas PR-957 treatment fully blocked the enhanced immunoproteasome activity caused by Ang II. Moreover, the administration of PR-957 significantly suppressed Ang II-induced cardiac hypertrophy, fibrosis, and inflammation. Mechanistically, PR-957 treatment inhibited phosphatase and tensin homolog on chromosome ten (PTEN) degradation, thereby inhibiting multiple signals including AKT/mTOR, ERK1/2, transforming growth factor-β, and IKB/NF-kB. Furthermore, PTEN blocking by its specific inhibitor VO-OHpic markedly attenuated the inhibitory effect of PR-957 on Ang II-induced cardiac hypertrophy in mice. We conclude that PR-957 blocks PTEN degradation and activates its downstream mediators, thereby attenuating Ang II-induced cardiac hypertrophy. These findings highlight that PR-957 may be a potential therapeutic agent for Ang II-induced hypertrophic remodeling.

The pathogenesis of cardiac hypertrophy is tightly associated with activation of intracellular hypertrophic signalling pathways, which leads to the synthesis of various proteins. Tripartite motif 10 (TRIM10) is an E3 ligase with important functions in protein quality control. However, its role in cardiac hypertrophy was unclear. In this study, neonatal rat cardiomyocytes (NRCMs) and TRIM10-knockout mice were subjected to phenylephrine (PE) stimulation or transverse aortic constriction (TAC) to induce cardiac hypertrophy in vitro and in vivo, respectively. Trim10 expression was significantly increased in hypertrophied murine hearts and PE-stimulated NRCMs. Knockdown of TRIM10 in NRCMs alleviated PE-induced changes in the size of cardiomyocytes and hypertrophy gene expression, whereas TRIM10 overexpression aggravated these changes. These results were further verified in TRIM10-knockout mice. Mechanistically, we found that TRIM10 knockout or knockdown decreased AKT phosphorylation. Furthermore, we found that TRIM10 knockout or knockdown increased ubiquitination of phosphatase and tensin homolog (PTEN), which negatively regulated AKT activation. The results of this study reveal the involvement of TRIM10 in pathological cardiac hypertrophy, which may occur by prompting of PTEN ubiquitination and subsequent activation of AKT signalling. Therefore, TRIM10 may be a promising target for treatment of cardiac hypertrophy.

T-cells play an important role in promoting mucosal immunity against pathogens, but the mechanistic basis for their homeostasis in the intestine is still poorly understood. We report here that T-cell-specific deletion of mTOR results in dramatically decreased CD4 and CD8 T-cell numbers in the lamina propria of both small and large intestines under both steady-state and inflammatory conditions. These defects result in defective host resistance against a murine enteropathogen, Citrobacter rodentium, leading to the death of the animals. We further demonstrated that mTOR deficiency reduces the generation of gut-homing effector T-cells in both mesenteric lymph nodes and Peyer's patches without obviously affecting expression of gut-homing molecules on those effector T-cells. Using mice with T-cell-specific ablation of Raptor/mTORC1 or Rictor/mTORC2, we revealed that both mTORC1 and, to a lesser extent, mTORC2 contribute to both CD4 and CD8 T-cell accumulation in the gastrointestinal (GI) tract. Additionally, mTORC1 but not mTORC2 plays an important role regulating the proliferative renewal of both CD4 and CD8 T-cells in the intestines. Our data thus reveal that mTOR is crucial for T-cell accumulation in the GI tract and for establishing local adaptive immunity against pathogens.

The aim of this study was to evaluate the ability of a 41-gene signature derived from breast cancer stem cells (BCSCs) to estimate the risk of metastasis and survival in breast cancer patients.