Ultrasound-Targeted Microbubble Destruction-Mediated miR-206 Overexpression Promotes Apoptosis and Inhibits Metastasis of Hepatocellular Carcinoma Cells Via Targeting PPIB.

BACKGROUND: Ultrasound-targeted microbubble destruction (UTMD) has been found to be an effective method for delivering microRNAs (miRNAs, miRs). The current study is aimed at discovering the potential anti-cancer effects of UTMD-mediated miR-206 on HCC. METHODS: In our study, the expressions of miR-206 and peptidyl-prolyl cis-trans isomerase B (PPIB) in HCC tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). PPIB expressions in HCC and adjacent normal tissues were analyzed by gene expression profiling interactive analysis (GEPIA). MiR-206 mimic and mimic control were transfected into HCC cells using UTMD. Potential binding sites between miR-206 and PPIB were predicted and confirmed by TargetScan and dual-luciferase reporter assay, respectively. Cell migration, invasion, and apoptosis were detected by wound healing assay, Transwell, and flow cytometry, respectively. The expressions of apoptosis-related proteins (Bax, Bcl-2), Epithelial-to-mesenchymal (EMT) markers (E-cadherin, N-cadherin and Snail) and PPIB were measured by Western blot. RESULTS: MiR-206 expression was downregulated while PPIB expression was upregulated in HCC, and PPIB was recognized as a target gene of miR-206 in HCC tissues. UTMD-mediated miR-206 inhibited HCC cell migration and invasion while promoting apoptosis via regulating the expressions of proteins related to apoptosis, migration, and invasion by targeting PPIB. CONCLUSION: Our results suggested that the delivery of UTMD-mediated miR-206 could be a potential therapeutic method for HCC treatment, given its effects on inhibiting cell migration and invasion and promoting cell apoptosis.

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence.

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

Following nature's roadmap: folding factors from plasma cells led to improvements in antibody secretion in S. cerevisiae.

Therapeutic protein production in yeast is a reality in industry with an untapped potential to expand to more complex proteins, such as full-length antibodies. Despite numerous engineering approaches, cellular limitations are preventing the use of Saccharomyces cerevisiae as the titers of recombinant antibodies are currently not competitive. Instead of a host specific approach, the possibility of adopting the features from native producers of antibodies, plasma cells, to improve antibody production in yeast. A subset of mammalian folding factors upregulated in plasma cells for expression in yeast and screened for beneficial effects on antibody secretion using a high-throughput ELISA platform was selected. Co-expression of the mammalian chaperone BiP, the co-chaperone GRP170, or the peptidyl-prolyl isomerase FKBP2, with the antibody improved specific product yields up to two-fold. By comparing strains expressing FKBP2 or the yeast PPIase Cpr5p, the authors demonstrate that speeding up peptidyl-prolyl isomerization by upregulation of catalyzing enzymes is a key factor to improve antibody titers in yeast. The findings show that following the route of plasma cells can improve product titers and contribute to developing an alternative yeast-based antibody factory.

Production of low-expressing recombinant cationic biopolymers with high purity.

The growing complexity of recombinant biopolymers for delivery of bioactive agents requires the ability to control the biomaterial structure with high degree of precision. Genetic engineering techniques have provided this opportunity to synthesize biomaterials in an organism such as E. coli with full control over their lengths and sequences. One class of such biopolymers is recombinant cationic biopolymers with applications in gene delivery, regenerative medicine and variety of other biomedical applications. Unfortunately, due to their highly cationic nature and complex structure, their production in E. coli expression system is marred by low expression yield which in turn complicates the possibility of obtaining pure biopolymer. SlyD and ArnA endogenous E. coli proteins are considered the major culprits that copurify with the low-expressing biopolymers during the metal affinity chromatography. Here, we compared the impact of different parameters such as the choice of expression hosts as well as metal affinity columns in order to identify the most effective approach in obtaining highly pure recombinant cationic biopolymers with acceptable yield. The results of this study showed that by using E. coli BL21(DE3) LOBSTR strain and in combination with our developed stringent expression and Ni-NTA purification protocols highly pure products in one purification step (>99% purity) can be obtained. This approach could be applied to the production of other complex and potentially toxic biopolymers with wide range of applications in biomedicine.

Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm.

Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together ß-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process. Here, we demonstrate that inclusion of the helper plasmid pTUM4, which results in the expression of four chaperones, DsbA, DsbC, FkpA, and SurA, increased yield on average 3.5-fold for the nine multi-disulfide bond single domain antibodies evaluated. No increase in production was observed for single domain antibodies containing only the canonical disulfide bond.

[Construction and Expression of Vector with mip/flaA Advantages Epitope Genes of Legionella pneumophila].

OBJECTIVE: To generate and express fusion vector with mip/flaA advantages epitope genes of Legionella pneumophila by select mip and flaA advantages epitope genes for future research on Legionella pneumophila protein vaccine. METHODS: Following analysis of secondary structure and surface properties such as: physical and chemical properties, hydropathy, plasticity, antigen index and extracellular domain of Mip and FlaA proteins by bioinformatics methods, the region which active epitope may exist was selected as advantages epitope region. Then, the recombinant plasmid pET-mip, pET-flaA and pET-mip/flaA with advantages epitope genes were constructed by PCR amplification and T4 ligase connection, and induced the expression in E. coli. RESULTS: Many potential antigenic epitopes in Mip and FlaA were identified, and the selected advantages epitope regions were cloned and expressed successfully. Moreover, the mip/flaA two advantages associated epitope fusion proteins were also successfully expressed. CONCLUSION: DNA Star software and Expasy online analysis system can successfully predict antigenic epitopes for Legionella pneumophila Mip and FlaA. And prokaryotic expression vector pET-mip/flaA with advantages epitope genes has been successfully constructed and efficiently expressed.

Periplasmic quality control in biogenesis of outer membrane proteins.

The ß-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

The Rab2A GTPase promotes breast cancer stem cells and tumorigenesis via Erk signaling activation.

Proline-directed phosphorylation is regulated by the prolyl isomerase Pin1, which plays a fundamental role in driving breast cancer stem-like cells (BCSCs). Rab2A is a small GTPase critical for vesicle trafficking. Here, we show that Pin1 increases Rab2A transcription to promote BCSC expansion and tumorigenesis in vitro and in vivo. Mechanistically, Rab2A directly interacts with and prevents dephosphorylation/inactivation of Erk1/2 by the MKP3 phosphatase, resulting in Zeb1 upregulation and ß-catenin nuclear translocation. In cancer cells, Rab2A is activated via gene amplification, mutation or Pin1 overexpression. Rab2A overexpression or mutation endows BCSC traits to primary normal human breast epithelial cells, whereas silencing Rab2A potently inhibits the expansion and tumorigenesis of freshly isolated BCSCs. Finally, Rab2A overexpression correlates with poor clinical outcome in breast cancer patients. Thus, Pin1/Rab2A/Erk drives BCSC expansion and tumorigenicity, suggesting potential drug targets.

Antioxidant activity is required for the protective effects of cyclophilin A against oxidative stress.

Cyclophilin (Cyp) belongs to a group of proteins that have peptidyl-prolyl cis-trans isomerase (PPIase) activity. CypA is the major cellular target for the immunosuppressive drug cyclosporin A and mediates its actions. Previous studies have demonstrated that CypA has diverse cellular functions and have suggested that CypA may function as an antioxidant. The present study investigated the antioxidant activity of CypA and its association with PPIase activity. The purified CypA/wild-type (WT) and CypA/P16S mutant proteins were active in PPIase assays. A total antioxidant capacity assay revealed that the purified CypA/WT protein had significantly higher antioxidant activity, whereas the CypA/P16S mutant was defective in its antioxidant activity. To confirm the importance of CypA antioxidant activity, CypA/P16S was overexpressed in Chang human liver cells and the rate of cell death was measured following treatment with cisplatin or H2O2. Overexpression of CypA/WT protected the cells against cisplatin or H2O2-induced oxidative damage, however, the CypA/P16S mutant had no effect. These findings suggested that CypA exhibits a protective antioxidant effect.

Propofol protects against angiotensin II-induced mouse hippocampal HT22 cells apoptosis via inhibition of p66Shc mitochondrial translocation.

Hippocampal neuronal oxidative stress and apoptosis have been reported to be involved in cognitive impairment, and angiotensin II could induce hippocampal oxidative stress and apoptosis. Propofol is a widely used intravenous anesthetic agent in clinical practice, and it demonstrates significant neuroprotective activities. In this study, we investigated the mechanism how propofol protected mouse hippocampal HT22 cells against angiotensin II-induced oxidative stress and apoptosis. Cell viability was evaluated with CCK8 kit. Protein expressions of active caspase 3, cytochrome c, p66(Shc), p-p66(shc)-Ser(36), protein kinase C ßII (PKCßII), Pin-1 and phosphatase A2 (PP2A) were measured by Western blot. Superoxide anion (O2(.-)) accumulation was measured with the reduction of ferricytochrome c. Compared with the control group, angiotensin II up-regulated expression of PKCßII, Pin-1 and PP2A, induced p66(Shc)-Ser(36) phosphorylation, and facilitated p66(Shc) mitochondrial translocation, resulting in O2(.-) accumulation, mitochondrial cytochrome c release, caspase 3 activation, and the inhibition of cell viability. Importantly, we found propofol inhibited angiotensin II-induced PKCßII and PP2A expression and improved p66(Shc) mitochondrial translocation, O2(.-) accumulation, mitochondrial cytochrome c release, caspase 3 activation, inhibition of cell viability. On the other hand, propofol had no effects on angiotensin II-induced Pin-1 expression and p66(Shc)-Ser(36) phosphorylation. Moreover, the protective effects of propofol on angiotensin II-induced HT22 apoptosis were similar with calyculin A, an inhibitor of PP2A and CGP53353, an inhibitor of PKCßII. However, the protective effect of propofol could be reversed by FTY720, an activator of PP2A, rather than PMA, an activator of PKCßII. Our data indicated that propofol down-regulated PP2A expression, inhibiting dephosphorylation of p66(Shc)-Ser(36) and p66(Shc) mitochondrial translocation, decreasing O2(.-) accumulation, reducing mitochondrial cytochrome c release, inhibiting caspase 3 activation. By these mechanisms, it protects mouse hippocampal HT22 cells against angiotensin II-induced apoptosis.

Pin1 positively affects tumorigenesis of esophageal squamous cell carcinoma and correlates with poor survival of patients.

BACKGROUND: Pin1 promotes oncogenesis by regulating multiple oncogenic signaling. In this study, we investigated the involvement of Pin1 in tumor progression and in the prognosis of human esophageal squamous cell carcinoma (ESCC). RESULTS: We observed that proliferation, clonogenicity and tumorigenesis of CE81T cells were inhibited by Pin1 knockdown. We next analyzed Pin1 expression in clinical ESCC specimens. When compared to the corresponding non-tumor part, Pin1 protein and mRNA levels in tumor part were higher in 84% and 62% patients, respectively. By immunohistochemistry, we identified that high Pin1 expression was associated with higher primary tumor stage (p = 0.035), higher overall cancer stage (p = 0.047) and poor overall survival (p < 0.001). Furthermore, the association between expression of Pin1 and levels of ß-catenin and cyclin D in cell line and clinical specimens was evaluated. ß-catenin and cyclin D1 were decreased in CE81T cells with Pin1 knockdown. Cyclin D1 level correlated with Pin1 expression in clinical ESCC specimens. CONCLUSIONS: Pin1 upregulation was associated with advanced stage and poor prognosis of ESCC. Pin1 knockdown inhibited aggressiveness of ESCC cells. ß-catenin and cyclin D1 were positively regulated by Pin1. These results indicated that targeting Pin1 pathway could represent a potential modality for treating ESCC.

Expression, purification and characterization of soluble recombinant peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum.

Vibrio anguillarum, a causative agent of vibriosis in finfish, crustaceans, and bivalves, is a Gram-negative, motile marine bacterium. Most bacteria have developed survival strategies in various environments. The aim of this study was to investigate the changes in protein expression of V. anguillarum O1 incubated under different conditions using two dimensional electrophoresis and MALDI-TOF MS/MS analysis. Result indicated that peptidyl-prolyl cis/trans isomerase (PPIase) expression was increasingly appeared when incubated at low temperature (15°C) and alkaline conditions (pH 10). Subsequently, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli to characterize the biochemical properties. The cloned ppi gene encoded 206 amino acids containing the conserved regions identified in FK506 binding pocket. To determine the optimal conditions of the purified recombinant PPIase protein (VaFKBP22), we used Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate and the highest enzymatic activity was found at 5°C and pH 6. VaFKBP22 was detected in the cytoplasm and periplasm of V. anguillarum O1. In addition, VaFKBP22 also showed chaperone activity and did not show cytotoxic activity.

Prolyl isomerase Pin1 acts downstream of miR200c to promote cancer stem-like cell traits in breast cancer.

Breast cancer stem-like cells (BCSC) have been implicated in tumor growth, metastasis, drug resistance, and relapse but druggable targets in appropriate subsets of this cell population have yet to be identified. Here we identify a fundamental role for the prolyl isomerase Pin1 in driving BCSC expansion, invasiveness, and tumorigenicity, defining it as a key target of miR200c, which is known to be a critical regulator in BCSC. Pin1 overexpression expanded the growth and tumorigenicity of BCSC and triggered epithelial-mesenchymal transition. Conversely, genetic or pharmacological inhibition of Pin1 reduced the abundance and self-renewal activity of BCSC. Moreover, moderate overexpression of miR200c-resistant Pin1 rescued the BCSC defect in miR200c-expressing cells. Genetic deletion of Pin1 also decreased the abundance and repopulating capability of normal mouse mammary stem cells. In human cells, freshly isolated from reduction mammoplasty tissues, Pin1 overexpression endowed BCSC traits to normal breast epithelial cells, expanding both luminal and basal/myoepithelial lineages in these cells. In contrast, Pin1 silencing in primary breast cancer cells freshly isolated from clinical samples inhibited the expansion, self-renewal activity, and tumorigenesis of BCSC in vitro and in vivo. Overall, our work demonstrated that Pin1 is a pivotal regulator acting downstream of miR200c to drive BCSC and breast tumorigenicity, highlighting a new therapeutic target to eradicate BCSC.

Involvement of peptidyl-prolyl isomerase Pin1 in the inhibitory effect of fluvastatin on endothelin-1-induced cardiomyocyte hypertrophy.

AIMS: Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions. MAIN METHODS: We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [(3)H]-leucine incorporation. KEY FINDINGS: Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [(3)H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin. SIGNIFICANCE: This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.

Helicobacter pylori secreted peptidyl prolyl cis, trans-isomerase drives Th17 inflammation in gastric adenocarcinoma.

Helicobacter pylori infection is characterized by an inflammatory infiltrate, consisting mainly of neutrophils and T cells. This study was undertaken to evaluate the type of gastric T cell response elicited by the secreted peptidyl prolyl cis, trans-isomerase of H. pylori (HP0175) in patients with distal gastric adenocarcinoma. The cytokine profile and the effector functions of gastric tumor-infiltrating lymphocytes (TILs) specific for HP0175 was investigated in 20 patients with distal gastric adenocarcinoma and H. pylori infection. The helper function of HP0175-specific TILs for monocyte MMP-2, MMP-9, and VEGF production was also investigated. TILs cells from H. pylori infected patients with distal gastric adenocarcinoma produced Interleukin (IL)-17 and IL-21 in response to HP0175. HP0175-specific TILs showed poor cytolytic activity while expressing helper activity for monocyte MMP-2, MMP-9 and VEGF production. These findings indicate that HP0175 is able to drive gastric Th17 response. Thus, HP0175, by promoting pro-inflammatory low cytotoxic TIL response, matrix degradation and pro-angiogenic pathways, may provide a link between H. pylori and gastric cancer.

Differential regulation of cellular senescence and differentiation by prolyl isomerase Pin1 in cardiac progenitor cells.

Autologous c-kit(+) cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing differentiation. The prolyl isomerase Pin1 regulates multiple signaling cascades by modulating protein folding and thereby activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 is expressed in CPCs in vitro and in vivo and is associated with increased proliferation. Pin1 is required for cell cycle progression and loss of Pin1 causes cell cycle arrest in the G1 phase in CPCs, concomitantly associated with decreased expression of Cyclins D and B and increased expression of cell cycle inhibitors p53 and retinoblastoma (Rb). Pin1 deletion increases cellular senescence but not differentiation or cell death of CPCs. Pin1 is required for endogenous CPC response as Pin1 knock-out mice have a reduced number of proliferating CPCs after ischemic challenge. Pin1 overexpression also impairs proliferation and causes G2/M phase cell cycle arrest with concurrent down-regulation of Cyclin B, p53, and Rb. Additionally, Pin1 overexpression inhibits replicative senescence, increases differentiation, and inhibits cell death of CPCs, indicating that cell cycle arrest caused by Pin1 overexpression is a consequence of differentiation and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve differentiation, and antagonize senescence.

Regulation of the microRNA 200b (miRNA-200b) by transcriptional regulators PEA3 and ELK-1 protein affects expression of Pin1 protein to control anoikis.

MicroRNA (miRNA) 200s regulate E-cadherin by directly targeting ZEB1/ZEB2, which are transcriptional repressors of E-cadherin. Decreased expression of E-cadherin results in cancer cells losing interaction with the extracellular matrix and detaching from the primary tumor. Normally, cells will undergo anoikis after losing interaction with the extracellular matrix. Cancer cells must, therefore, possess the ability to resist anoikis during the process of metastasis. Here we show that miRNA-200b regulates anoikis by directly targeting the 3' UTR of Pin1 mRNA and regulating Pin1 expression at the translational level. We found that down-regulation of miRNA-200b promotes cancer cells survival during metastasis, and the homeless state of these cells resulted in decreased expression of miRNA-200b in the MCF-7 cell line. We also found that expression of miRNA-200b is down-regulated in human breast cancer during lymph node metastasis, which has a significant negative correlation with Pin1 expression. Two members of the ETS (E-26) family (PEA3 and ELK-1) regulate the expression of miRNA-200b. PEA3 promotes the expression of miRNA-200b, and ELK-1 is a transcriptional repressor of miRNA-200b. In addition, miRNA-200b regulates the activity of PEA3 and ELK-1 via the Pin1-pERK pathway and forms self-regulated feedback loops. This study characterizes the role of miRNA-200b in the regulation of anoikis and demonstrates the regulation of its own expression in the process of metastasis.

Proteomic analysis for testis of mice exposed to carbon ion radiation.

This paper investigates the mechanism of action of heavy ion radiation (HIR) on mouse testes. The testes of male mice subjected to whole body irradiation with carbon ion beam (0.5 and 4Gy) were analyzed at 7days after irradiation. A two-dimensional gel electrophoresis approach was employed to investigate the alteration of protein expression in the testes. Spot detection and matching were performed using the PDQuest 8.0 software. A difference of more than threefold in protein quantity (normalized spot volume) is the standard for detecting differentially expressed protein spots. A total of 11 differentially expressed proteins were found. Protein identification was performed using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF). Nine specific proteins were identified by searching the protein sequence database of the National Center for Biotechnology Information. These proteins were found involved in molecular chaperones, metabolic enzymes, oxidative stress, sperm function, and spermatogenic cell proliferation. HIR decreased glutathione activity and increased malondialdehyde content in the testes. Given that Pin1 is related to the cell cycle and that proliferation is affected by spermatogenesis, we analyzed testicular histological changes and Pin1 protein expression through immunoblotting and immunofluorescence. Alterations of multiple pathways may be associated with HIR toxicity to the testes. Our findings are essential for studies on the development, biology, and pathology of mouse testes after HIR in space or radiotherapy.

Pin1-Nanog expression in human glioma is correlated with advanced tumor progression.

The stemness gene Nanog has been shown to play an important role in tumor development, including glioma. Nanog is phosphorylated at multiple Ser/Thr-Pro motifs, which promotes the interaction between Nanog and the prolyl isomerase Pin1, leading to Nanog stabilization by suppressing its ubiquitination. The present study investigated the expression and relationship of Pin1 and Nanog in human gliomas. Significantly higher mRNA and protein expression levels of Pin1 and Nanog were demonstrated in 120 glioma specimens of different pathological grades by RT-PCR, immunohistochemistry staining and western blot analysis. The relative levels of Pin1 expression, as well as Nanog expression, were significantly positively correlated with pathological grade. Moreover, a positive correlation of Pin1 and Nanog expression in human gliomas was noted. Co-localization of Pin1 and Nanog was observed in the perinuclear space in the cytoplasm of glioma cells detected by immunofluorescence staining. Significantly positive correlation between Pin1 and Nanog in gliomas indicated that Pin1 and Nanog may be related to tumorigenesis and development of glioma cells.

Pathogen-free screening of bacteria-specific hybridomas for selecting high-quality monoclonal antibodies against pathogen bacteria as illustrated for Legionella pneumophila.

Antibodies are potent biological tools increasingly used as detection, diagnostic and therapeutic reagents. Many technological advances have optimized and facilitated production and screening of monoclonal antibodies. We report here an original method to screen for antibodies targeting biosafety level 2 or 3 pathogens without the fastidious handling inherent to pathogen use. A double ELISA screening was performed using as coated antigen transformed Escherichia coli expressing at its surface a protein specific to the pathogenic bacteria versus control untransformed E. coli. This method was applied to Legionella, using the surface-exposed Mip protein (macrophage infectivity potentiator). This screening proved to be an excellent means of selecting mAbs that bind Legionella pneumophila 1 surface-exposed Mip protein. This method also appears more biologically relevant than screening using the recombinant Mip protein alone and less tedious than a test performed directly on Legionella bacteria. We obtained 21 mAbs that bind strongly to L. pneumophila serogroups 1 to 13, and we validated their use in a rapid ELISA (performed in 4.5 h) and an immunochromatographic test (20 min).