Mechanosensitive brain tumor cells construct blood-tumor barrier to mask chemosensitivity

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Model membrane platforms to study protein-membrane interactions

Abstract Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.     

Model membrane platforms to study protein-membrane interactions

Abstract

Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.     

The tumor microenvironment and its contribution to tumor evolution toward metastasis

Cancer cells acquire cell-autonomous capacities to undergo limitless proliferation and survival through the activation of oncogenes and inactivation of tumor suppressor genes. Nevertheless, the formation of a clinically relevant tumor requires support from the surrounding normal stroma, also referred to as the tumor microenvironment. Carcinoma-associated fibroblasts, leukocytes, bone marrow-derived cells, blood and lymphatic vascular endothelial cells present within the tumor microenvironment contribute to tumor progression. Recent evidence indicates that the microenvironment provides essential cues to the maintenance of cancer stem cells/cancer initiating cells and to promote the seeding of cancer cells at metastatic sites. Furthermore, inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize host immune response toward specific phenotypes impacting tumor progression. A growing number of studies demonstrate a positive correlation between angiogenesis, carcinoma-associated fibroblasts, and inflammatory infiltrating cells and poor outcome, thereby emphasizing the clinical relevance of the tumor microenvironment to aggressive tumor progression. Thus, the dynamic and reciprocal interactions between tumor cells and cells of the tumor microenvironment orchestrate events critical to tumor evolution toward metastasis, and many cellular and molecular elements of the microenvironment are emerging as attractive targets for therapeutic strategies.     

Rapid chemosensitivity assay with human normal and tumor cells in vitro

Summary Neutral red assay, as an index of cytotoxicity, has been applied to predictive screening of chemotherapeutic agents. Human hepatoma and melanoma tumor cells and normal melanocytes, keratinocytes and fibroblasts were incubated for 2, 24, and 48 h with graded concentrations of cis-platinum (0.1 to 80 μM), doxorubicin (0.01 to 100 μM), and 5-fluorouracil (1 to 1000 μM). Cells were most sensitive after 48 h. Tumor cells, based on 50% toxicity values, were 2–4 times more sensitive than the normal cells, except for cis-platinum, where only melanoma cells, as compared to normal melanocytes, showed a marked difference in cytotoxic response. Methotrexate (1 to 10 μM) toxicity could be reversed in the presence of 100 μM of leucovorin. This sensitive, rapid, and economical assay is suitable for preclinical screening and drug development. This work has been supported, in part, by funds from Schering Corporation, New Jersey, and Chevron Environmental Health Center, Inc., California.     

Caveolin-1 is a negative regulator of tumor growth in glioblastoma and modulates chemosensitivity to temozolomide

Caveolin-1 (Cav-1) is a critical regulator of tumor progression in a variety of cancers where it has been shown to act as either a tumor suppressor or tumor promoter. In glioblastoma multiforme, it has been previously demonstrated to function as a putative tumor suppressor. Our studies here, using the human glioblastoma-derived cell line U-87MG, further support the role of Cav-1 as a negative regulator of tumor growth. Using a lentiviral transduction approach, we were able to stably overexpress Cav-1 in U-87MG cells. Gene expression microarray analyses demonstrated significant enrichment in gene signatures corresponding to downregulation of MAPK, PI3K/AKT and mTOR signaling, as well as activation of apoptotic pathways in Cav-1-overexpressing U-87MG cells. These same gene signatures were later confirmed at the protein level in vitro. To explore the ability of Cav-1 to regulate tumor growth in vivo, we further show that Cav-1-overexpressing U-87MG cells display reduced tumorigenicity in an ectopic xenograft mouse model, with marked hypoactivation of MAPK and PI3K/mTOR pathways. Finally, we demonstrate that Cav-1 overexpression confers sensitivity to the most commonly used chemotherapy for glioblastoma, temozolomide. In conclusion, Cav-1 negatively regulates key cell growth and survival pathways and may be an effective biomarker for predicting response to chemotherapy in glioblastoma.     

Engineering platforms for directed evolution of Laccase from Pycnoporus cinnabarinus

While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the α-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved α-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k(cat). While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (～23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).     

The relationship between the in vitro chemosensitivity of tumor cells and tumor response in vivo in an experimental tumor model

Cell lines derived from a panel of five histologically distinct murine adenocarcinomas of the colon (MAC) were used to assess whether or not a colony-forming assay could have retrospectively predicted the wide range of in vivo responses to chlorambucil (CHL). The predictive value of the clonogenic assay was significantly improved when fractions (one-tenth) of the plasma drug AUC (from the area under the drug clearance curves); were used to determine clonogenic cell kill in vitro, instead of one-tenth peak plasma drug concentration and total plasma drug AUC exposures. Despite the good correlation between in vitro and in vivo responses observed, the clonogenic assay could not forecast the site-dependent response of MAC 15A to CHL. These site-dependent responses cannot be explained in terms of the inherent sensitivity of tumor cells themselves, suggesting that caution must be applied in the interpretation of in vitro chemosensitivity assays.     

Tracking tumor evolution via prostate-specific antigen: an individual post-operative study

Background  

The progress of the prostate-specific antigen (PSA) level after radical prostatectomy is observed for a patient in order to extract information about the mode of tumor cell growth. Although PSA values are determined routinely to find the doubling time of the prostate marker, to our knowledge, this analysis is the first in the literature.     

microRNA‐577 suppresses tumor growth and enhances chemosensitivity in colorectal cancer

In this work, we aimed to determine the expression and biological functions of microRNA (miR)‐577 in colorectal cancer (CRC). The results showed that miR‐577 was downregulated in CRC specimens and cell lines. Restoration of miR‐577 significantly suppressed the proliferation and colony formation and induced a G0/G1 cell cycle arrest in CRC cells. 5‐Fluorouracil (5‐FU)‐resistant SW480 cells (SW480/5‐FU) were found to have elevated levels of miR‐577. Ectopic expression of miR‐577 enhanced 5‐FU sensitivity in SW480/5‐FU cells. Heat shock protein 27 (HSP27) was identified as a target gene of miR‐577. Enforced expression of HSP27 reversed the effects of miR‐577 on CRC cell growth and 5‐FU sensitivity. Xenograft tumors derived from miR‐577‐overexpressing SW480 cells exhibited significantly slower growth than control tumors. In conclusion, our results support that miR‐577 acts as a tumor suppressor in CRC likely through targeting HSP27. Therefore, miR‐577 may have therapeutic potential in the treatment of CRC.     

Postconfluency MDCK monolayers as an in vitro model of solid tumor chemosensitivity

Shortly after reaching confluency, canine MDCK cells enter a prolonged state of basal growth with doubling times of 200–300 hours. These values are similar to those commonly exhibited by in vivo solid tumors at clinically relevant sizes. By comparison with rapidly growing sparse density cultures, the postconfluent monolayers displayed a pronounced resistance to deazauridine, deoxyspergualin, and 5-fluorouridine. Drug concentrations required for unit levels of effect increased from several fold to several orders of magnitude as cells entered high density basal growth. This high density chemoresistance was observed for both growth inhibition and cytotoxicity, but was much more pronounced with the former. Dose-response curves were biphasic, suggesting that growth inhibition and cytotoxicity may be mediated by different mechanisms of drug action. The pronounced chemoresistance of postconfluent MDCK monolayers is similar to that encountered with many clinical solid neoplasms. It suggests that postconfluency monolayers, like multicellular spheroids and cellular multilayers, may provide better in vitro models of solid tumor chemosensitivity than subconfluent monolayer and suspension cultures.Abbreviations DAUR 3-deazauridine - DSG deoxyspergualin - 5-FUrd 5-fluorouridine - HDCR high-density chemoresistance - IC50 concentration producing 50% inhibition of growth - LC50 concentration producing 50% loss of cell protein     

转录组平台技术及其在代谢工程中的应用

组学技术在系统水平上对细胞代谢进行全面的分析,极大地促进了代谢工程的发展和应用。全基因组水平的转录分析可以使研究者更加精确地评估细胞表型,加深对细胞代谢的理解。而且转录组分析也有助于研究者鉴定菌种改良的目标基因,加速对微生物细胞工厂的合理设计及构建。文中介绍了3种主要转录组平台技术的原理,并总结了转录组学在代谢工程领域中应用的最新进展和未来发展趋势。     

Transmission-recovery trade-offs to study parasite evolution

Parasite evolution is mainly studied through a trade-off involving host death (i.e., virulence) and transmission. In addition to the lack of evidence, this trade-off largely fails to understand the evolution of sublethal parasite effects. Here, I argue that considering host recovery as a main selection pressure faced by the parasite helps to address these problems and opens new perspectives for the study of parasite evolution. Using an embedded model, I show how a trade-off between transmission and recovery may emerge from within-host dynamics if immune activation is assumed to depend on the parasite's overall growth rate. I also show that the value of the parasite's optimal growth rate strongly depends on the immunological state of the host. Transmission-recovery trade-offs are of particular interest to the study of the evolution of human pathogens because of the use of antipathogen treatments, which strengthens the recovery constraint.     

Polynitroxyl hemoglobin: a pharmacokinetic study of covalently bound nitroxides to hemoglobin platforms

Adding antioxidant activities to hemoglobin-based oxygen carriers (HBOCs) represents a means of reducing cell-free hemoglobin-mediated oxidative cascades. We have covalently bound nitroxides, a class of antioxidant enzyme mimetics, to HBOCs. The objectives of this study were (1) to evaluate the pharmacokinetic (PK) effects of administering nitroxide covalently bound to HBOCs compared to those of free nitroxide coadministered with HBOCs and (2) to elucidate the effects of differing molecular weight HBOCs on the PK of bound nitroxide in a conscious guinea pig model of 25% blood exchange transfusion. Two HBOC platforms were used, intramolecular cross-linked hemoglobin (XLHb) and dextran polymerized/conjugated XLHb (PolyHb). Polynitroxylation was achieved by reacting 4-(2-bromoacetamido)-2,2,6,6,-tetramethylpiperidine-1-oxyl with XLHb or PolyHb to form polynitroxylated XLHb and polynitroxylated PolyHb, respectively, whereas a physical mixture of XLHb or PolyHb with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl was prepared to reflect a molar equivalence to HBOC-bound nitroxide. Plasma concentrations of two redox states, nitroxide and hydroxylamine, were determined by electron paramagnetic resonance spectroscopy. Results are presented to illustrate the influence of covalent labeling and HBOC molecular weight on nitroxide PK. The therapeutic potential of polynitroxylation of HBOCs as it relates to observations from the current and previously reported studies is discussed.     

Further study of the correlation between na-pump activity and membrane chemosensitivity

Using internally dialyzed neurons of Helix, we have examined the effects of sodium-pump activity and intracellular ATP concentration on transmembrane currents induced by acetylcholine (ACh) and gamma-aminobutyric acid (GABA). We also report on the effects of pump activity and levels of intracellular ATP on binding by Helix ganglia of 3H-alpha-bungarotoxin (3H-alpha-BT) and 3H-GABA. Both ouabain-containing and potassium-free solutions depressed the neurotransmitter-induced transmembrane current of one type of dialyzed neurons. An increase in the intracellular ATP concentration led to a depression of ACh-induced currents and to the disappearance of the blocking effect of ouabain on these currents. Intracellular ADP had a similar but smaller effect on transmitter-induced currents, and intracellular AMP was ineffective. The depressing effect of internal ATP on ACh-induced currents was absent in the presence of an inhibitor of membrane phosphorylation (dinitrophenol). The binding of tritium-labeled alpha-BT and GABA to the membranes was depressed by both ouabain-containing and K-free solutions and also by compounds (theophylline and NaF) which increase the levels of intracellular ATP. The results suggest that the Na pump modulates the affinity of ACh and GABA membrane receptors by the regulation of the phosphorylated state of membrane receptors.     

Introduction to cloud computing: platforms and solutions

This special issue of the cluster computing journal will feature articles that discuss tools and applications for cloud computing. Specifically, it aims at delivering the state-of-the-art research on current cloud computing tools topics, and at promoting the cloud applications discipline by bringing to the attention of the community novel problems that must be investigated.     

Protein evolution constraints and model-based techniques to study them

There have been substantial improvements in statistical tools for assessing the evolutionary roles of mutation and natural selection from interspecific sequence data. The importance of having the rate at which a point mutation occurs depend on the DNA sequence at sites surrounding the mutation is now better appreciated and can be accommodated in probabilistic models of protein evolution. To quantify the evolutionary impact of some aspect of phenotype, one promising strategy is to develop a system for predicting phenotype from the DNA sequence and to then infer how the evolutionary rates of sequence change are affected by the predicted phenotypic consequences of the changes. Although statistical tools for characterizing protein evolution are improving, the list of candidate phenomena that can affect rates of protein evolution is long and the relative contributions of these phenomena are only beginning to be disentangled.