The role of mTOR and phospho-p70S6K in pathogenesis and progression of gastric carcinomas: an immunohistochemical study on tissue microarray

<p>Abstract</p> <p>Background</p> <p>mTOR signaling pathway and its downstream serine/threonine kinase p70S6k were frequently activated in human cancers. The dysregulation of the mTOR pathway has been found to be a contributing factor of a variety of different cancer. To investigate the role of mTOR signal pathway in the stepwise development of gastric carcinomas, we analyzed the correlations between the mTOR and P70S6K expression and clinic pathological factors and studied its prognostic role in gastric carcinomas.</p> <p>Methods</p> <p>mTOR and phospho-p70S6K proteins were examined by immunohistochemistry on tissue microarray containing gastric carcinomas (n = 412), adenomas (n = 47) and non-neoplastic mucosa (NNM, n = 197) with a comparison of their expression with clinicopathological parameters of carcinomas.</p> <p>Results</p> <p>There was no difference of mTOR expression between these three tissues (p > 0.05). Cytoplasmic phospho(p)-P706SK was highly expressed in adenoma, compared with ANNMs (p < 0.05), whereas its nuclear expression was lower in gastric carcinomas than gastric adenoma and ANNMs (p < 0.05). These three markers were preferably expressed in the older patients with gastric cancer and intestinal-type carcinoma (p < 0.05). mTOR expression was positively correlated with the cytoplasmic and nuclear expression of p-P70S6K(p < 0.05). Nuclear P70S6K was inversely linked to tumor size, depth of invasion, lymph node metastasis and UICC staging (p < 0.05). Univariate analysis indicated that expression of mTOR and nuclear p-P70S6K was closely linked to favorable prognosis of the carcinoma patients (p < 0.05). Multivariate analysis showed that age, depth of invasion, lymphatic invasion, lymph node metastasis, Lauren's classification and mTOR expression were independent prognostic factors for overall gastric carcinomas (p < 0.05).</p> <p>Conclusion</p> <p>Aberrant expression of p-P70S6K possibly contributes to pathogenesis, growth, invasion and metastasis of gastric carcinomas. It was considered as a promising marker to indicate the aggressive behaviors and prognosis of gastric carcinomas.</p

High Yield Production Process for Shigella Outer Membrane Particles

Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30–45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from Gram-negative bacteria

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium

Prevention of surgical site infections in spine surgery: an international survey of clinical practices among expert spine surgeons

Study Design: Questionnaire-based survey.Objectives: Surgical site infection (SSI) is a common complication in spine surgery but universal guidelines for SSI prevention are lacking. The objectives of this study are to depict a global status quo on implemented prevention strategies in spine surgery, common themes of practice and determine key areas for future research.Methods: An 80-item survey was distributed among spine surgeons worldwide via email. The questionnaire was designed and approved by an International Consensus Group on spine SSI. Consensus was defined as more than 60% of participants agreeing to a specific prevention strategy.Results: Four hundred seventy-two surgeons participated in the survey. Screening for Staphylococcus aureus (SA) is not common, whereas preoperative decolonization is performed in almost half of all hospitals. Body mass index (BMI) was not important for surgery planning. In contrast, elevated HbAIc level and hypoalbuminemia were often considered as reasons to postpone surgery. Cefazoline is the common drug for antimicrobial prophylaxis. Alcohol-based chlorhexidine is mainly used for skin disinfection. Double-gloving, wound irrigation, and tissue-conserving surgical techniques are routine in the operating room (OR). Local antibiotic administration is not common. Wound closure techniques and postoperative wound dressing routines vary greatly between the participating institutions.Conclusions: With this study we provide an international overview on the heterogeneity of SSI prevention strategies in spine surgery. We demonstrated a large heterogeneity for pre-, peri- and postoperative measures to prevent SSI. Our data illustrated the need for developing universal guidelines and for testing areas of controversy in prospective clinical trails.Scientific Assessment and Innovation in Neurosurgical Treatment Strategie